Enzyme compositions for the improved enzymatic hydrolysis of cellulose and methods of using same

ABSTRACT

A process for the enzymatic hydrolysis of cellulose to produce a hydrolysis product comprising glucose from a pretreated lignocellulosic feedstock and enzymes for use in the process are provided. The process comprises hydrolyzing an aqueous slurry of a pretreated lignocellulosic feedstock with cellulase enzymes, one or more than one β-glucosidase enzyme and a binding agent for binding the β-glucosidase enzyme to fiber solids present in the aqueous slurry. During the hydrolysis, both the cellulase enzyme and β-glucosidase enzyme bind to the fiber solids. The hydrolysis is performed in a solids-retaining hydrolysis reactor so that unhydrolyzed fiber solids and bound enzyme are retained in the reactor longer than the aqueous phase of the slurry.

This application is a national stage application of PCT/CA2007/001133having an international filing date of Jun. 22, 2007, which claimsbenefit of U.S. provisional application No. 60/815,818 filed Jun. 22,2006, both of which are incorporated by reference herein.

FIELD OF INVENTION

The present invention relates to enzymes for the hydrolysis of celluloseand methods of using same. More specifically, the present inventionrelates to cellulase and β-glucosidase enzymes for the enzymatichydrolysis of cellulose to produce a hydrolysis product comprisingglucose from a pretreated lignocellulosic feedstock.

BACKGROUND OF THE INVENTION

Fuel ethanol is currently produced from feedstocks such as corn starch,sugar cane, and sugar beets. However, the potential for production ofethanol from these sources is limited as most of the farmland which issuitable for the production of these crops is already in use as a foodsource for humans. Furthermore, the production of ethanol from thesefeedstocks has a negative impact on the environment because fossil fuelsused in the conversion process produce carbon dioxide and otherbyproducts.

The production of ethanol from cellulose-containing feedstocks, such asagricultural wastes, grasses, and forestry wastes, has received muchattention in recent years. The reasons for this are because thesefeedstocks are widely available and inexpensive and their use forethanol production provides an alternative to burning or landfillinglignocellulosic waste materials. Moreover, a byproduct of celluloseconversion, lignin, can be used as a fuel to power the process insteadof fossil fuels. Several studies have concluded that, when the entireproduction and consumption cycle is taken into account, the use ofethanol produced from cellulose generates close to nil greenhouse gases.

The lignocellulosic feedstocks that are the most promising for ethanolproduction include (1) agricultural residues such as corn stover, wheatstraw, barley straw, oat straw, rice straw, canola straw, and soybeanstover; (2) grasses such as switch grass, miscanthus, cord grass, andreed canary grass; and (3) forestry wastes such as aspen wood andsawdust.

The first process step of converting lignocellulosic feedstock toethanol involves breaking down the fibrous material to liberate sugarmonomers, such as glucose, from the feedstock for conversion to ethanolin the subsequent step of fermentation. The two primary processes areacid hydrolysis, which involves the hydrolysis of the feedstock using asingle step of acid treatment, and enzymatic hydrolysis, which involvesan acid pretreatment followed by hydrolysis with cellulase enzymes.

In the acid hydrolysis process, the feedstock is subjected to steam andsulfuric acid at a temperature, acid concentration and length of timethat are sufficient to hydrolyze the cellulose to glucose and thehemicellulose to xylose and arabinose. The sulfuric acid can beconcentrated (25-80% w/w) or dilute (3-8% w/w). The glucose is thenfermented to ethanol using yeast, and the ethanol is recovered andpurified by distillation.

In the enzymatic hydrolysis process, the steam temperature, sulfuricacid concentration and treatment time are chosen to be milder than thatin the acid hydrolysis process such that the cellulose surface area isgreatly increased as the fibrous feedstock is converted to a muddytexture, but there is little conversion of the cellulose to glucose. Thepretreated cellulose is then hydrolyzed to glucose in a subsequent stepthat uses cellulase enzymes, and the steam/acid treatment in this caseis known as pretreatment. Prior to the addition of enzyme, the pH of theacidic feedstock is adjusted to a value that is suitable for theenzymatic hydrolysis reaction. Typically, this involves the addition ofalkali to a pH of between about 4 to about 6, which is the optimal pHrange for cellulases, although the pH can be higher if alkalophiliccellulases are used.

In one type of pretreatment process, the pressure produced by the steamis brought down rapidly with explosive decompression, which is known assteam explosion. Foody, (U.S. Pat. No. 4,461,648) describes theequipment and conditions used in steam explosion pretreatment. Steamexplosion with sulfuric acid added at a pH of 0.4 to 2.0 has been thestandard pretreatment process for two decades. It produces pretreatedmaterial that is uniform and requires less cellulase enzyme to hydrolyzecellulose than other pretreatment processes.

Cellulase enzymes catalyze the hydrolysis of the cellulose(β-1,4-D-glucan linkages) in the feedstock to products such as glucose,cellobiose, and other cellooligosaccharides. Cellulase is a generic termdenoting a multienzyme mixture comprising exo-cellobiohydrolases (CBH),endoglucanases (EG) and β-glucosidases (βG) that can be produced by anumber of plants and microorganisms. Cellulase enzymes worksynergistically to hydrolyze cellulose to glucose. CBHI and CBHIIgenerally act on the ends of the glucose polymers in cellulosemicrofibrils liberating cellobiose (Teeri and Koivula, Carbohydr.Europe, 1995, 12:28-33), while the endoglucanases act at randomlocations on the cellulose. Together, these enzymes hydrolyze celluloseto smaller cellooligosaccharides, primarily cellobiose. Cellobiose ishydrolyzed to glucose by β-glucosidase. It is known that mostexo-cellobiohydrolases (CBH) and endoglucanases (EG) bind to cellulosein the feedstock via carbohydrate-binding modules (CBMs), such ascellulose-binding domains (CBDs), while most β-glucosidase enzymes,including Trichoderma and Aspergillus β-glucosidase enzymes, do notcontain such binding modules and thus remain in solution. Cellulaseenzymes may contain a linker region that connects the catalytic domainto the carbohydrate binding module. The linker region is believed tofacilitate the activity of the catalytically active domain.

Cellulase enzymes containing a CBD have been produced by geneticengineering. For example, U.S. Pat. No. 5,763,254 (Wöldike et al.)discloses genetically engineered cellulose degrading enzymes derivedfrom Humicola, Fusarium and Myceliopthora containingcarbohydrate-binding domains. The goal of the studies was to producecellulose or hemicellulose-degrading enzymes with novel combinations ofthe catalytically active domain, the linker region and the CBD or toproduce CBD-containing cellulose or hemicellulose-degrading enzymes fromthose that lack a CBD. However, the ability of these novel enzymes tohydrolyze lignocellulosic feedstock was not demonstrated.

One significant problem with enzymatic hydrolysis processes is the largeamount of cellulase enzyme required, which increases the cost of theprocess. The cost of cellulase accounts for more than 50% of the cost ofhydrolysis. There are several factors that contribute to the enzymerequirement, but one of particular significance is the presence ofcompounds that reduce the reaction rate of cellulases and/ormicroorganisms in the subsequent fermentation of the sugar. For example,glucose released during the process inhibits cellulases, particularlyβ-glucosidase (Alfani et al., J. Membr. Sci., 1990, 52:339-350).Cellobiose produced during cellulose hydrolysis is a particularly potentinhibitor of cellulase (Tolan et al. in Biorefineries—IndustrialProcesses and Products, Vol. 1 Ed. Kamm et al., Chapter 9, page 203).Other soluble inhibitors are produced during pretreatment includingsugar degradation products such as furfural and hydroxyl-methylfurfural; furan derivatives; organic acids, such as acetic acid; andsoluble phenolic compounds derived from lignin. These compounds alsoinhibit yeast, which decreases ethanol production and consequently makesthe process more costly. Although the effects of inhibitors can bereduced by performing the hydrolysis at a more dilute concentration,this requires the use of a large hydrolysis reactor, which adds to theexpense of the process.

Simultaneous Saccharification and Fermentation (SSF) is a method ofconverting lignocellulosic biomass to ethanol which minimizes glucoseinhibition of cellulases (see for example Ghosh et al., Enzyme Microb.Technol., 1982, 4:425-430). In an SSF system, enzymatic hydrolysis iscarried out concurrently with yeast fermentation of glucose to ethanol.During SSF, the yeast removes glucose from the system by fermenting itto ethanol and this decreases inhibition of the cellulase. However, adisadvantage of this process is that the cellulase enzymes are inhibitedby ethanol. In addition, SSF is typically carried out at temperatures of35-38° C., which is lower than the 50° C. optimum for cellulase andhigher than the 28° C. optimum for yeast. This intermediate temperatureresults in substandard performance by both the cellulase enzymes and theyeast. Thus, the inefficient hydrolysis requires very long reactiontimes and very large reaction vessels, both of which are costly.

Another approach that has been proposed to reduce inhibition by glucose,cellobiose, and other soluble inhibitors is removing hydrolysis productsthroughout hydrolysis by carrying out the reaction in a membranereactor. A membrane reactor contains an ultrafiltration membrane whichretains particles and high molecular weight components, such as enzyme,while allowing lower molecular weight molecules, such as sugars, to passthrough the membrane as permeate.

An example of a process utilizing a membrane reactor is described inOhlson and Trägårdh (Biotech. Bioeng., 1984, 26:647-653), in which theenzymatic hydrolysis of pretreated sallow (a willow tree species) iscarried out in a reactor with a membrane having a 10,000 molecularweight cut off. Cellulases have a molecular weight of 50,000 and aretherefore retained by the membrane in the hydrolysis reactor, whilesugars are removed and replaced with buffer solution from a feedcontainer with fresh substrate added intermittently. The rate ofhydrolysis, as well as the yield of the soluble sugars, is enhanced dueto the removal of inhibitors. However, a disadvantage of such reactorsis that the membranes required for a commercial hydrolysis system areextremely large and expensive. The membranes are also prone to foulingby suspended solids present in the reaction mixture.

Various groups have investigated the recovery and recycling of cellulaseenzymes during enzymatic hydrolysis to reduce the amount of the enzymenecessary during the conversion process. In some cases, this has alsoinvolved the continuous removal of hydrolyzates from the reactionmixture, thereby removing inhibitory compounds.

For example, Ishihara et al. (Biotech. Bioeng., 1991, 37:948-954)disclose the recycling of cellulase enzymes during the hydrolysis ofsteamed hardwood and hardwood kraft pulp in a reactor. The processinvolves the removal of a cellulase reaction mixture from the reactor,followed by the removal of insoluble residue containing lignin from themixture by filtering with suction. The cellulase enzymes that are in thefiltrate are separated from hydrolysis products, such as glucose andcellobiose, by ultrafiltration and then returned to the hydrolysisreactor. As stated by the investigators, a disadvantage of this systemis that the extra step of solids removal would be impractical in anindustrial application due to the rise in the cost of raw material. Inaddition, most of the cellulases remain bound to the cellulose and aredifficult to recover.

Larry et al. (Appl. Microbiol. Biotechnol., 1986, 25:256-261) describean approach for the re-use of cellulases which involves performing thehydrolysis in a column reactor containing cellulose (Solka Floc). Thehydrolyzed sugars are continuously removed by percolating the columnwith a steady stream of buffer. According to the investigators, theremoval of sugar products should reduce product inhibition and enhancehydrolysis efficiencies. However, inadequate hydrolysis is obtainedsince unbound β-glucosidase and endoglucanase elute from the column.

Knutsen and Davis (Appl. Biochem. Biotech., 2002, 98-100:1161-1172)report a combined inclined sedimentation and ultrafiltration process forrecovering cellulase enzymes during the hydrolysis of lignocellulosicbiomass. The goal of the process is to remove larger lignocellulosicparticles so a membrane filter does not become clogged during asubsequent step of ultrafiltration. The process first involves treatinglignocellulosic particles with cellulase enzymes and then feeding theresulting mixture into an inclined settler. Large lignocellulosicparticles, including enzyme bound to the particles, are retained in theinclined settler, while smaller particles and soluble enzyme are carriedout with the settler overflow. The overflow is then fed to a crossflowultrafiltration unit to recover unbound cellulases, while allowing forthe passage of sugars. After ultrafiltration, the recovered cellulasesare added to the hydrolysis reactor. The lignocellulosic particlesremaining in the inclined settler, along with the bound enzyme, arereturned to the reactor along with the settler underflow. Onedisadvantage of this system is that the operation of such a system onthe scale of a commercial hydrolysis reactor, which is likely to beabout 70 feet tall and process thousands of gallons of slurry everyhour, will be prohibitively difficult. A second disadvantage of thissystem is that the concentration of glucose and cellobiose in thereactor remains unchanged throughout the process so that a high level ofinhibition still occurs. A further disadvantage of the process is thatit requires an expensive ultrafiltration step to recover unboundcellulases.

Mores et al. (Appl. Biochem. Biotech., 2001, 91-93:297-309) report acombined inclined sedimentation and ultrafiltration process similar tothat described by Knutsen and Davis (supra). However, the process ofMores et al. involves an extra clarification step involving subjectingthe settler overflow to microfiltration prior to ultrafiltration toreduce fouling of the ultrafiltration membrane. The process of Mores etal. would be subject to the same limitations as those described forKnutsen and Davis (supra).

U.S. Pat. No. 3,972,775 (Wilke et al.) reports a process for recyclingcellulase in which the hydrolysis products are separated into an aqueoussugar-containing phase and a solid phase containing unhydrolyzed spentsolids after the hydrolysis is complete. The spent solids are washedwith water to recover enzyme adsorbed on it and the resulting wash watercontaining the desorbed enzyme is fed to the hydrolysis reaction. Theremaining spent solids can be used as a source of fuel for the system.However, the process of Wilke et al. incurs the cost of the additionalwater wash after the hydrolysis, which is significant due to the largeamount of solid material and the fine particulate nature of the solids.In addition, the process does not result in the removal of inhibitors ofcellulase enzymes present during the hydrolysis reaction since theseparation of hydrolyzates is carried out after completion of thehydrolysis reaction.

Ramos et al. (Enzyme Microb. Technol., 1993, 15:19-25) disclose aprocess in which steam-exploded eucalyptus chips are hydrolyzed usingcellulase with removal of soluble sugars and the recycling of enzyme.The process involves stopping the reaction at selected incubation timesand collecting the unhydrolyzed, enzyme-containing residue on a sinteredglass filter. The enzyme-containing residue is washed with hydrolysisbuffer to remove soluble sugars. The washed residue is then re-suspendedin fresh hydrolysis buffer containing fresh β-glucosidase enzyme andincubated at 45° C. for subsequent hydrolysis. A problem with thisprocess is that the repeated addition of fresh β-glucosidase afterre-suspension would significantly increase the expense of the process.

Lee et al. (Biotech. Bioeng., 1994, 45:328-336) examine the recycling ofcellulase enzymes in a procedure involving over five successive roundsof hydrolysis. The process involves adding cellulase enzymes andβ-glucosidase (Novozym® 188) to peroxide-treated birch and recoveringthe residual substrate by filtering after 12 hours of hydrolysis. Freshsubstrate is then added to the recovered residual substrate to achieve atotal substrate concentration of 2% and the resulting mixture isre-suspended in buffer containing β-glucosidase and the hydrolysis isallowed to continue. Cellulase recycling followed by hydrolysis issubsequently repeated three times. Also disclosed is a procedure forrecycling cellulases present in the complete reaction mixture bothbefore and after all the cellulose is hydrolyzed. Similar to Ramos etal., a limitation of this process is that β-glucosidase must be added tothe reaction at each recycling step.

U.S. Pat. No. 5,962,289 (Kilbum et al.) discloses a three-step enzymatichydrolysis. The first step of the process involves adding bothendoglucanase and exoglucanase to a lignocellulosic material to behydrolyzed to cellobiose. The second step involves adding this materialto an Avicel® column to adsorb the endoglucanase and exoglucanase. In athird step, the eluent containing cellobiose is then applied to a secondAvicel® column containing β-glucosidase immobilized via a CBD. Theimmobilized β-glucosidase hydrolyzes the cellobiose into glucose. Onelimitation of this method is that the production of glucose is carriedout in three distinct process steps, which is highly complex and costly.A second limitation is that sending the slurry of partially-hydrolyzedlignocellulosic material through the column of Avicel® at a high flowrate typical of a commercial hydrolysis process is very difficult. Inaddition, the highly inhibitory effects of cellobiose are present duringthe cellulose hydrolysis.

At present, there is much difficulty in the art to operate an efficientenzymatic hydrolysis of cellulose. A key obstacle is overcoming theinhibitory effects of glucose and especially cellobiose to cellulase.The development of such a system remains a critical requirement for aprocess to convert cellulose to glucose.

SUMMARY OF THE INVENTION

The present invention relates to enzymes for the hydrolysis of celluloseand methods of using same. More specifically, the present inventionrelates to cellulase and β-glucosidase enzymes for the enzymatichydrolysis of cellulose to produce a hydrolysis product comprisingglucose from a pretreated lignocellulosic feedstock.

It is an object of the invention to provide an improved method for thetreatment of lignocellulosic feedstocks.

According to the present invention, there is provided an enzymecomposition for the enzymatic hydrolysis of cellulose to produce ahydrolysis product comprising glucose from a pretreated lignocellulosicfeedstock, the enzyme composition comprising cellulase enzymes, one ormore than one β-glucosidase enzyme and a binding agent for binding theβ-glucosidase enzyme to the pretreated lignocellulosic feedstock,wherein the hydrolysis is carried out by:

(i) providing an aqueous slurry of the pretreated lignocellulosicfeedstock, said aqueous slurry comprising fiber solids and an aqueousphase;

(ii) hydrolyzing the aqueous slurry with the enzyme compositioncomprising cellulase enzymes, one or more than one β-glucosidase enzymeand the binding agent in a solids-retaining hydrolysis reactor toproduce said hydrolysis product comprising glucose, wherein thecellulase enzymes and the one or more than one β-glucosidase enzyme bindto the fiber solids; and

(iii) withdrawing unhydrolyzed fiber solids and the aqueous phase whichcomprises the hydrolysis product from the solids-retaining hydrolysisreactor,

wherein the unhydrolyzed fiber solids are retained in thesolids-retaining hydrolysis reactor for about 6 hours to about 148 hourslonger than the aqueous phase.

The binding agent may be a carbohydrate-binding module operably linkedto the one or more than one β-glucosidase enzyme. Preferably, thecarbohydrate-binding module is a cellulose-binding domain.

The present invention also pertains to the enzyme composition asdescribed above, wherein the cellulase enzymes are produced byAspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or acombination thereof. Preferably, the cellulase enzymes are produced byTrichoderma.

The present invention also pertains to the enzyme composition asdescribed above, wherein the cellulase enzymes comprise acellobiohydrolase (CBH) selected from the group consisting of CBHI andCBHII cellulase enzymes, and combinations thereof, and an endoglucanase(EG) selected from the group consisting of EGI, EGII, EGIV, EGV and EGVIcellulase enzymes, and combinations thereof.

The present invention also pertains to the enzyme composition asdescribed above, wherein about 75% to about 100% (w/w) of the totalcellulase enzymes present in the enzyme composition bind to the fibersolids in step (ii).

The present invention also pertains to the enzyme composition asdescribed above, wherein about 75% to about 100% (w/w), or about 90% toabout 100% (w/w), of the total β-glucosidase enzyme present in theenzyme composition comprises a cellulose-binding domain. Thecellulose-binding domain may be a Family I cellulose-binding domain.Furthermore, the cellulose-binding domain may be a bacterial or fungalcellulose-binding domain. Optionally, the β-glucosidase enzyme comprisesa linker, which operably links the cellulose-binding domain to theβ-glucosidase enzyme.

The present invention also pertains to the enzyme composition asdescribed above, wherein the one or more than one β-glucosidase enzymeis produced by Aspergillus, Humicola, Trichoderma, Bacillus,Thermobifida, or a combination thereof. Preferably the β-glucosidaseenzyme is produced by Trichoderma or Aspergillus. The β-glucosidaseenzyme may be naturally occurring or a genetically modified fusionprotein.

The present invention also pertains to the enzyme composition asdescribed above, further comprising separating the unhydrolyzed solidsfrom the hydrolysis product comprising glucose to produce separatedsolids and an aqueous solution comprising glucose. The separated solidsmay be re-suspended in an aqueous solution, and the hydrolysiscontinued.

According to the present invention, there is also provided a use of anenzyme composition for the enzymatic hydrolysis of cellulose to producea hydrolysis product comprising glucose from a pretreatedlignocellulosic feedstock, the enzyme composition comprising cellulaseenzymes, one or more than one β-glucosidase enzyme and a binding agentfor binding the β-glucosidase enzyme to the pretreated lignocellulosicfeedstock, wherein the use of the enzyme composition comprises:

(i) providing an aqueous slurry of the pretreated lignocellulosicfeedstock, said aqueous slurry comprising fiber solids and an aqueousphase;

(ii) hydrolyzing the aqueous slurry with the enzyme compositioncomprising cellulase enzymes, one or more than one β-glucosidase enzymeand the binding agent in a solids-retaining hydrolysis reactor toproduce said hydrolysis product comprising glucose, wherein thecellulase enzymes and the one or more than one β-glucosidase enzyme bindto the fiber solids; and

(iii) withdrawing unhydrolyzed fiber solids and the aqueous phase, whichcomprises the hydrolysis product, from the solids-retaining hydrolysisreactor, wherein the unhydrolyzed fiber solids are retained in thesolids-retaining hydrolysis reactor for about 6 hours to about 148 hourslonger than the aqueous phase. The binding agent may be acarbohydrate-binding module operably linked to the one or more than oneβ-glucosidase enzyme. Preferably, the carbohydrate-binding module is acellulose-binding domain.

The present invention also pertains to use of the enzyme composition asdescribed above, wherein the solids-retaining hydrolysis reactor isoperated as a batch or a continuous reactor. The present invention alsopertains to the use of the enzyme composition as described above,wherein the solids-retaining hydrolysis reactor is part of a hydrolysissystem that comprises two or more hydrolysis reactors and the hydrolysisreactors are operated in series, in parallel, or a combination thereof.

The present invention also pertains to the use of the enzyme compositionas described above, wherein the solids-retaining hydrolysis reactor is asettling reactor and wherein, during the step of hydrolyzing (step(ii)), at least a portion of the fiber solids settle in the settlingreactor. The settling reactor may be a tower with the aqueous slurryflowing upward through the tower.

The present invention also pertains to the use of the enzyme compositionas described above, wherein the cellulase enzymes are produced byAspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or acombination thereof. Preferably, the cellulase enzymes are produced byTrichoderma.

The present invention also pertains to the use of the enzyme compositionas described above, wherein the cellulase enzymes comprise acellobiohydrolase (CBH) selected from the group consisting of CBHI andCBHII cellulase enzymes, and combinations thereof, and an endoglucanase(EG) selected from the group consisting of EGI, EGII, EGIV, EGV and EGVIcellulase enzymes, and combinations thereof.

The present invention also pertains to the use of the enzyme compositionas described above, wherein between about 75% and about 100% (w/w) ofthe total cellulase enzymes present in the enzyme composition bind tothe fiber solids in step (ii).

The present invention also pertains to the use of the enzyme compositionas described above, wherein about 75% to about 100% (w/w), preferablyabout 90% to about 100% (w/w), of the total β-glucosidase enzyme presentin the enzyme composition comprises a cellulose-binding domain. Thecellulose-binding domain may be a Family I cellulose-binding domain.Furthermore, the cellulose-binding domain may be a bacterial or fungalcellulose-binding domain. Optionally, the β-glucosidase enzyme comprisesa linker.

The present invention also pertains to the use of the enzyme compositionas described above, wherein the β-glucosidase enzyme is produced byAspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or acombination thereof. Preferably the β-glucosidase enzyme is produced byTrichoderma or Aspergillus. The β-glucosidase enzyme may be naturallyoccurring or a genetically modified fusion protein. The β-glucosidaseenzyme may be native to the host, or may be native to another genus orspecies and inserted into the host to be expressed.

The present invention also pertains to the use of the enzyme compositionas described above, further comprising separating the unhydrolyzedsolids from the hydrolysis product comprising glucose to produceseparated solids and an aqueous solution comprising glucose. Theseparated solids may then be re-suspended in an aqueous solution, andthe hydrolysis continued in a second hydrolysis.

According to the present invention, there is also provided a process forthe enzymatic hydrolysis of cellulose to produce a hydrolysis productcomprising glucose from a pretreated lignocellulosic feedstock, theprocess comprising:

(i) providing an aqueous slurry of the pretreated lignocellulosicfeedstock, said aqueous slurry comprising fiber solids and an aqueousphase;

(ii) hydrolyzing the aqueous slurry with cellulase enzymes, one or morethan one β-glucosidase enzyme and a binding agent for binding theβ-glucosidase enzyme to the fiber solids in a solids-retaininghydrolysis reactor to produce said hydrolysis product comprisingglucose, wherein the cellulase enzymes and the one or more than oneglucosidase enzyme bind to the fiber solids; and

(iii) withdrawing unhydrolyzed fiber solids and the aqueous phase whichcomprises the hydrolysis product from the solids-retaining hydrolysisreactor, wherein the unhydrolyzed fiber solids are retained in thesolids-retaining hydrolysis reactor for about 6 hours to about 148 hourslonger than the aqueous phase.

The binding agent may be a carbohydrate binding module operably linkedto the one or more than one β-glucosidase enzyme. Preferably, thecarbohydrate binding module is a cellulose-binding domain.

The present invention also pertains to the process as described above,wherein the one or more than one β-glucosidase enzyme comprises acellulose-binding domain that binds to cellulose in the pretreatedfeedstock.

The present invention also pertains to the process as described above,wherein, in the step of providing (step (i)), the aqueous slurry has asuspended or undissolved solids content of about 3% to about 30% (w/w).This aqueous slurry may be concentrated prior to the step of hydrolyzing(step (ii)). Preferably, the aqueous slurry is prepared in water.

The present invention also pertains to the process as described above,wherein the unhydrolyzed solids are separated from the hydrolysisproduct comprising glucose to produce separated solids and a firstaqueous solution comprising glucose. The separated solids may bere-suspended in an aqueous solution, and the hydrolysis continued in asecond hydrolysis to produce a second aqueous solution comprisingglucose and unhydrolyzed solids. The first and second aqueous solutionscomprising glucose may be combined to produce a combined sugar stream.The sugar stream may be fermented to produce a fermentation brothcomprising ethanol.

The present invention also pertains to the process as described above,wherein the unhydrolyzed solids are separated from the hydrolysisproduct by microfiltration, centrifugation, vacuum filtration orpressure filtration. Preferably, the unhydrolyzed solids are separatedfrom the glucose by microfiltration.

The present invention also pertains to the process as described above,wherein the solids-retaining hydrolysis reactor is part of a hydrolysissystem comprising two or more hydrolysis reactors and the hydrolysisreactors are operated in series, in parallel, or a combination thereof.The hydrolysis system may comprise a hydrolysis reactor selected fromthe group consisting of an agitated tank, an unmixed tank, an agitatedtower and an unmixed tower. The agitated tower or the unmixed tower maybe either a downflow tower or an upflow tower. The process may be a acontinuous process with continuous feeding of the aqueous slurry andcontinuous withdrawal of the hydrolyzed solids and the aqueous phase ora batch process.

The present invention also pertains to the process as described above,wherein the re-suspended slurry has a solids content of between about10% and 15% (w/w). The aqueous solution for re-suspending the separatedsolids may be process water.

The pretreated lignocellulosic feedstock may be obtained from wheatstraw, oat straw, barley straw, corn stover, soybean stover, canolastraw, rice straw, sugar cane, bagasse, switch grass, reed canary grass,cord grass, or miscanthus.

The present invention also pertains to the enzyme composition, use ofthe enzyme composition, or process as described above, wherein, in thestep of hydrolyzing (step (ii)), the cellulase enzymes are added at adosage of about 1.0 to about 40.0 IU per gram of cellulose. Thecellulase enzymes may be produced by Aspergillus, Humicola, Trichoderma,Bacillus, Thermobifida, or a combination thereof. Preferably, betweenabout 75% and 100% (w/w) of the total cellulase enzymes present bind tothe fiber solids in step (ii).

The present invention also pertains to the process as described above,wherein, in the step of hydrolyzing (step (ii)), the one or more thanone β-glucosidase enzyme is added at a dosage of about 35 to about 200IU per gram of cellulose. The β-glucosidase enzymes may be produced byAspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or acombination thereof. Preferably, the β-glucosidase is produced byAspergillus or Trichoderma. The β-glucosidase enzyme may be native tothe host, or may be native to another genus or species and inserted intothe host to be expressed.

The present invention also pertains to the process as described above,wherein, in the step of hydrolyzing (step (ii)), the pH of the aqueousslurry is from about 4.5 to about 5.5, or between about 4.5 and 5.0. Thetemperature of the aqueous slurry may be between about 45° C. to about55° C. The aqueous slurry may be prepared in water. If the unhydrolyzedsolids are re-suspended, the aqueous solution used for re-suspension maybe process water.

The present invention also pertains to the process as described above,wherein the step of hydrolyzing (step (ii)) is carried out for a totalresidence time of the aqueous phase of about 12 to about 200 hours. Thestep of continuing the hydrolysis of the re-suspended slurry may becarried out for about 12 to about 200 hours.

According to the present invention, there is also provided a use of anenzyme composition in the enzymatic hydrolysis of cellulose to produce ahydrolysis product comprising glucose from a pretreated lignocellulosicfeedstock, the enzyme composition comprising cellulase enzymes, one ormore than one β-glucosidase enzyme and a binding agent for binding theβ-glucosidase enzyme to the pretreated lignocellulosic feedstock,wherein said enzymatic hydrolysis is performed in a solids-retaininghydrolysis reactor.

The present invention overcomes several disadvantages of the prior artby taking into account the difficulties encountered in steps carried outduring the conversion of lignocellulosic feedstock to glucose. Asdescribed herein, cellulase enzymes, one or more than one β-glucosidaseenzyme and a binding agent for binding the β-glucosidase enzyme to thelignocellulosic feedstock are used to enzymatically hydrolyze an aqueousslurry of a pretreated lignocellulosic feedstock. In one embodiment ofthe invention, the enzymatic hydrolysis is carried out in asolids-retaining hydrolysis reactor so that fiber solids in the aqueousslurry are retained in the hydrolysis reactor longer than the aqueousphase of the slurry to increase the reaction time between the cellulasesand cellulose. The fiber solids are retained in the hydrolysis reactorby settling, filtration, centrifugation, or other means that partiallyor totally separate the aqueous phase from the fiber solids. In aconventional hydrolysis, the β-glucosidase is not bound to thecellulose, and thus would be removed from the hydrolysis reactor withthe aqueous phase. The removal of β-glucosidase would cause theaccumulation of cellobiose, which is inhibitory to cellulase, andsignificantly decreases the rate of cellulose hydrolysis. By contrast,in practicing the invention, the β-glucosidase enzyme remains bound tocellulose in the fiber solids, and is not removed from the hydrolysisreactor with the aqueous phase. With β-glucosidase remaining in thehydrolysis reactor, cellobiose is converted to glucose and does notaccumulate in the reactor. The combination of the longer contact timebetween cellulase and cellulose, and the conversion of cellobiose toglucose, results in a hydrolysis system with improved efficiency and adecreased cellulase requirement.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1A is a process flow diagram illustrating the steps of processingthe lignocellulosic feedstock according to embodiments of the invention.FIG. 1B is a process flow diagram illustrating the steps of processingthe lignocellulosic feedstock using upflow hydrolysis reactors.

FIGS. 2A and 2B show the hydrolysis of 5% pretreated wheat strawcellulose by Trichoderma cellulase containing β-glucosidase with a CBDwith and without resuspension. The hydrolysis with resuspension wasfiltered and re-suspended at 24 hours, while the hydrolysis withoutresuspension was run undisturbed. In FIG. 2A, the cellulase dosage is 16mg/g and in FIG. 2B, the cellulase dosage is 24 mg/g.

FIG. 3 shows the hydrolysis of 5% pretreated wheat straw cellulose byTrichoderma cellulase containing native β-glucosidase which lacks a CBD.The hydrolyses were filtered and re-suspended at 24 hr.

FIGS. 4A and 4B are SDS-PAGE gels of purified β-glucosidase without aCBD (βG) and β-glucosidase with a CBD (βG-CBD) after incubation in thepresence (+) or absence (−) of pretreated wheat straw. In FIG. 4A, theincubation was carried out at 4° C. and in FIG. 4B, the incubation wascarried out at 50° C. After 30 minutes of incubation, the reactionmixtures were centrifuged and the supernatant fraction separated bySDS-PAGE and visualized by Coomassie Blue stain.

DETAILED DESCRIPTION

The following description is of preferred embodiments.

The present invention relates to enzymes for the improved hydrolysis ofcellulose. More specifically, the present invention relates tocellulases and β-glucosidase enzymes for the improved enzymaticconversion of lignocellulosic feedstocks and methods of using same.

The following description is of an embodiment by way of example only andwithout limitation to the combination of features necessary for carryingthe invention into effect.

There is provided an enzyme composition and method for processinglignocellulosic feedstocks which improves the efficiency of the process.The process involves performing a hydrolysis of a pretreated feedstockslurry with cellulases and one or more than one β-glucosidase that bindsto the pretreated feedstock via a binding agent, and retaining theunhydrolyzed fiber solids, which comprise lignin, cellulose, and otherinsoluble compounds in the hydrolysis reactor longer than the aqueousphase, which comprises glucose, glucose oligomers and cellobiose. Thefiber solids are retained by carrying out the hydrolysis in asolids-retaining hydrolysis reactor, including, but not limited to asettling reactor. By retaining the fiber solids in the hydrolysisreactor, the contact time between the enzyme and cellulose is increased,thereby increasing the degree of cellulose conversion.

After the hydrolysis, the unhydrolyzed fiber solids and an aqueous phasewhich comprises hydrolysis product are withdrawn from the hydrolysisreactor. The unhydrolyzed fiber solids may then be separated from thehydrolysis product to produce separated fiber solids and an aqueoussolution comprising glucose. This step is optional, but is oftendesirable because it facilitates fermentation of the sugar and furtherprocessing of the unhydrolyzed solids. The cellulases and the boundβ-glucosidase enzyme remain with the separated fiber solids by virtue oftheir ability to bind to the solids. In one embodiment of the invention,the hydrolysis of the separated fiber solids may then be continued.Separating the unhydrolyzed fiber solids from the hydrolysis productresults in the removal of glucose, cellobiose, and other solubleinhibitors, or their concentrations are reduced, so that the hydrolysiscan continue without, or with reduced, inhibition by these compounds.

The process may be a continuous process, with continuous feeding ofpretreated feedstock slurry and withdrawal of hydrolysis product.Alternately, the process may be a batch process.

The process is carried out on a pretreated feedstock slurry so that thedigestibility of the cellulose in the feedstock by the cellulase enzymesis enhanced. The cellulase enzymes convert at least a portion of thecellulose in the feedstock to glucose, cellobiose, glucose oligomers, ora combination thereof.

The feedstock for use in the practice of the invention is alignocellulosic material. By the term “lignocellulosic feedstock”, it ismeant any type of plant biomass such as, but not limited to, plantbiomass of cultivated crops such as, but not limited to, grasses, forexample, but not limited to C4 grasses, such as switch grass, cordgrass, rye grass, miscanthus, reed canary grass, or a combinationthereof, or sugar processing residues such as baggase, or beet pulp,agricultural residues, for example, soybean stover, corn stover, ricestraw, rice hulls, barley straw, corn cobs, wheat straw, canola straw,rice straw, oat straw, oat hulls, corn fiber, or a combination thereof,or forestry wastes such as recycled wood pulp fiber, sawdust, hardwood,for example, aspen wood and sawdust, softwood, or a combination thereof.Furthermore, the lignocellulosic feedstock may comprise otherlignocellulosic waste material such as, but not limited to, newsprint,cardboard and the like. Lignocellulosic feedstock may comprise onespecies of fiber or alternatively, lignocellulosic feedstock maycomprise a mixture of fibers that originate from differentlignocellulosic feedstocks. In addition, the lignocellulosic feedstockmay comprise fresh lignocellulosic feedstock, partially driedlignocellulosic feedstock, fully dried lignocellulosic feedstock or acombination thereof.

Lignocellulosic feedstocks comprise cellulose in an amount greater thanabout 20%, more preferably greater than about 30%, more preferablygreater than about 40% (w/w), still more preferably greater than 50%(w/w). For example, the lignocellulosic feedstock may comprise fromabout 20% to about 50% (w/w) cellulose, or more, or any amounttherebetween, for example, but not limited to 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48 and 50% (w/w) cellulose. Thelignocellulosic feedstock also comprises lignin in an amount greaterthan about 10%, more preferably in an amount greater than about 15%(w/w). The lignocellulosic feedstock may also comprise a combined amountof sucrose, fructose and starch in an amount less than about 20%,preferably less than about 10% (w/w). The weight percentages disclosedabove are relative to the mass of the lignocellulosic feedstock as itexists prior to pretreatment.

The preferred lignocellulosic feedstocks include (1) agriculturalresidues such as corn stover, wheat straw, barley straw, oat straw, ricestraw, canola straw, and soybean stover; and (2) grasses such as switchgrass, miscanthus, cord grass and reed canary grass.

The present invention is practiced with lignocellulosic material thathas been pretreated. Pretreatment methods are intended to deliver asufficient combination of mechanical and chemical action so as todisrupt the fiber structure and increase the surface area of feedstockaccessible to cellulase enzymes. Mechanical action typically includes,but is not limited to, the use of pressure, grinding, milling,agitation, shredding, compression/expansion, or other types ofmechanical action. Chemical action can include, but is not limited to,the use of heat (often steam), acid, alkali and solvents. Severalchemical and mechanical pretreatment methods are well known in the art.

Prior to pretreatment, the lignocellulosic feedstock may be leached.This may be carried out, for example, as disclosed in WO 02/070753(Griffin et al., which is incorporated herein by reference). However,even if leaching is practiced, a substantial amount of inhibitingcompounds are produced in the subsequent pretreatment process.

The pretreatment is employed to increase the susceptibility of thelignocellulosic feedstock slurry to hydrolysis by cellulase enzymes. Forexample, the pretreatment may be carried out to hydrolyze thehemicellulose, or a portion thereof, that is present in thelignocellulosic feedstock to monomeric sugars, for example xylose,arabinose, mannose, galactose, or a combination thereof. Preferably, thepretreatment is performed so that nearly complete hydrolysis of thehemicellulose and a small amount of conversion of cellulose to glucoseoccurs. The cellulose is hydrolyzed to glucose in a subsequent step thatuses cellulase enzymes. During the pretreatment, typically a diluteacid, from about 0.02% (w/v) to about 2% (w/v), or any amounttherebetween, is used for the pretreatment of the lignocellulosicfeedstock. Preferably, the pretreatment is carried out at a temperatureof about 180° C. to about 250° C. for a time of about 6 seconds to about120 seconds, at a pH of about 0.8 to about 2.0. Pretreatment may becarried out in a single stage or in more than one stage. Preferably, atleast one stage is carried out at the temperature range, for the timeperiod and the pH range set out above.

One approach to the pretreatment of the feedstock is steam explosion,using the process conditions described in U.S. Pat. Nos. 4,461,648 and4,237,226 (which are herein incorporated by reference). Another methodof pretreating the feedstock slurry involves continuous pretreatment,meaning that the lignocellulosic feedstock is pumped through a reactorcontinuously. Continuous acid pretreatment is familiar to those skilledin the art, see for example U.S. Pat. No. 5,536,325 (Brink); co-pendingU.S. application No. U.S. 60/687,224 (Foody and Tolan); U.S. Pat. No.4,237,226 (Grethlein; which are incorporated herein by reference). Othermethods that are known within the art may be used as required for thepreparation of a pretreated feedstock, for example, but not limited to,those disclosed in U.S. Pat. No. 4,556,430 (Converse et al.; which isincorporated herein by reference).

The pretreated lignocellulosic feedstock may optionally be washed withwater prior to enzymatic hydrolysis. The washing or leaching step canremove some of the inhibitors of cellulase enzymes and yeast, such asdissolved sugars and sugar degradation products, dissolved lignin andphenolic compounds and other organic compounds in the system. However,although washing after pretreatment falls within the scope of theinvention, it suffers from the disadvantage of being expensive and thatit is difficult to remove all of the insoluble impurities present.

The pretreated lignocellulosic material is slurried in an aqueoussolution to produce an aqueous feedstock slurry or “aqueous slurry”. Forexample, but without wishing to be limiting, the aqueous solution may beprocess water, fresh water, steam condensate or process recycle streams.The concentration of pretreated lignocellulosic feedstock in the aqueousslurry depends on the particle size, water retention, pump capacity andother properties of the feedstock. Typically, the concentration isbetween about 3% and 30% (w/w), or between about 10% and about 20% (w/w)fiber solids (also known as suspended or undissolved solids), or anyamount therebetween. The aqueous slurry preferably has a solidsconcentration that enables it to be pumped. As is well known in the art,the concentration of suspended or undissolved solids can be determinedby filtering a sample of the slurry using glass microfiber filter paper,washing the filter cake with water, and drying the cake overnight at105° C. It is preferred that the fiber solids comprise at least about20% to about 70% cellulose by weight, or any amount therebetween. Forexample, the suspended solids may comprise 30%, 35%, 40%, 45%, 50%, 55%,60%, 65% or 70% cellulose by weight.

The pH of the aqueous slurry is generally adjusted to within the rangeof the optimum pH for the cellulase enzymes used. Generally, the pH ofthe aqueous slurry is adjusted to within a range of about 3.0 to about7.0, or any pH therebetween. Typically, the pH is within a range ofabout 4.5 to about 5.5, or any pH therebetween. However, it should beappreciated that the pH of the slurry can be higher or lower than about4.5 to about 5.5 if the cellulase enzymes used are alkalophilic oracidophilic. The pH of the slurry may be adjusted using any suitableacid or base known in the art. For example, if the slurry is basic(e.g., if a basic pretreatment is performed), sulfuric acid may be used.If the slurry is acidic, the pH may be adjusted with bases selected fromthe group consisting of ammonia, ammonium hydroxide, lime, calciumhydroxide, potassium hydroxide, magnesium hydroxide, sodium hydroxideand a mixture thereof. Preferably, the base is selected from the groupconsisting of ammonia, ammonium hydroxide and sodium hydroxide.

The temperature of the aqueous feedstock slurry is adjusted so that itis within the optimum range for the activity of the cellulase enzymes.Generally, a temperature of about 45° C. to about 55° C., or anytemperature therebetween, is suitable for most cellulase enzymes.However, the temperature of the slurry may be higher for thermophiliccellulase enzymes.

The cellulase enzymes and a β-glucosidase enzyme with binding agent areadded to the aqueous slurry, prior to, during, or after the adjustmentof the temperature and pH of the aqueous slurry. Preferably thecellulase enzymes and the β-glucosidase enzyme are added to thepretreated lignocellulosic feedstock slurry after the adjustment of thetemperature and pH of the slurry. The hydrolysis of the pretreatedlignocellulosic material is then carried out.

A cellulase is an enzyme with hydrolytic activity toward cellulose inthe fiber solids and that comprises at least one catalytic domain. Acellulase enzyme generally has additional domains, including, but notlimited to a carbohydrate-binding module or other functional domains.

By the term “cellulase enzymes” or “cellulases,” it is meant a mixtureof enzymes that hydrolyze cellulose. The mixture may includeglucobiohydrolases (GBH), cellobiohydrolases (CBH) and endoglucanases(EG). Although GBH enzymes may form a component of the enzyme mixture,their use in the enzymatic hydrolysis of cellulose is less common thanCBH and EG enzymes. In a non-limiting example, the mixture includes CBHand EG enzymes. The GBH enzyme primarily hydrolyzes cellulose polymerchains from their ends to release glucose, while the CBH enzymeprimarily hydrolyzes cellulose polymer chains from their ends to releasecellobiose and the EG enzyme primarily hydrolyzes cellulose polymer inthe middle of the chain. The GBH enzyme may be an enzyme having anactivity of type EC#3.2.1.73, the CBH enzyme may have an enzyme activityof type EC#3.2.1.91 and the EG enzyme may have an activity of typeEC#3.2.1.4 or EC#3.2.1.151.

The cellulase enzymes can be produced by a number of plants andmicroorganisms. The process of the present invention can be carried outwith any type of cellulase enzymes, regardless of their source. Amongthe most widely studied, characterized and commercially producedcellulases are those obtained from fungi of the genera Aspergillus,Humicola, and Trichoderma, and from the bacteria of the genera Bacillusand Thermobifida. Cellulase produced by the filamentous fungiTrichoderma longibrachiatum comprises at least two cellobiohydrolaseenzymes termed CBHI and CBHII and at least four EG enzymes. As well,EGI, EGII, EGIII, EG V and EGVI cellulases have been isolated fromHumicola insolens (see Schulein et al., Proceedings of the Second TRICELSymposium on Trichoderma reesei Cellulases and Other Hydrolases, Espoo1993, P. Suominen and T. Reinikainen, Eds. Foundation for Biotechnicaland Industrial Fermentation Research, Helsinki 8:109-116, which isincorporated herein by reference).

The CBHI enzyme is defined as a CBH that primarily hydrolyzes cellulosepolymer chains by a retaining mechanism as would be known to one ofskill in the art. The CBHI enzyme may be processive. The CBHI enzyme maybe a member of a Family 7, 10 or Family 48 glycohydrolases. In apreferred embodiment, the CBHI enzyme is a member of Family 7. In a morepreferred embodiment, the CBHI enzyme is the Family 7 CBHI fromTrichoderma.

The CBHII enzyme is defined as an enzyme that primarily hydrolyzescellulose polymer chains by an inverting mechanism as would be known toone of skill in the art. The CBHII enzyme may be processive. The CBHIIenzyme may be a member of Family 6, 9 or 74. In a preferred embodiment,the CBHII enzyme is a member of Family 6. In a more preferredembodiment, the CBHII enzyme is the Family 6 CBHII from Trichoderma.

Examples of EG enzymes that may be used in the practice of thisinvention are set out in Table 1 below:

TABLE 1 Examples of EG enzymes Glucohydrolase EG enzyme Family EGI 7EGII 5 EGIII 12 EGIV 61 EGV 45 EGVI 74

Preferably, the EG enzymes are fungal enzymes, such as enzymes expressedfrom Trichoderma. The EG enzymes preferably contain a CBD (cellulosebinding domain), although a certain proportion of the EG enzymes may beincluded in the cellulase enzyme mixture that lack a CBD.

The cellulase enzyme dosage is chosen to convert the cellulose of thepretreated feedstock to glucose. For example, an appropriate cellulasedosage can be about 1.0 to about 40.0 Filter Paper Units (FPU or IU) pergram of cellulose, or any amount therebetween. The FPU is a standardmeasurement familiar to those skilled in the art and is defined andmeasured according to Ghose (Pure and Appl. Chem., 1987, 59:257-268).

Cellulase enzymes used in the practice of this invention bind tocomponents of the pretreated feedstock. However, it should be apparentthat the enzyme composition may comprise some cellulases that do notbind to the pretreated lignocellulosic feedstock, such as those that donot comprise a cellulose-binding domain. The percentage of cellulaseenzymes that bind to cellulose (solids) may be between about 75% and100% (w/w); for example, the percentage of cellulase enzymes that bindsto cellulose may be about 75, 78, 80, 83, 85, 87, 90, 93, 95, 97, or100% (w/w) of the total cellulase enzymes present in the enzymecomposition.

The conversion of cellobiose to glucose is carried out by theβ-glucosidase. By the term “β-glucosidase”, it is meant any enzyme thathydrolyzes the glucose dimer, cellobiose, to glucose. The activity ofthe β-glucosidase enzyme is defined by its activity by the EnzymeCommission as EC#3.2.1.21. The β-glucosidase enzymes for use in thisinvention are water soluble. There are many microbes that makeβ-glucosidase and the properties of these enzymes vary, includingstructure (molecular weight, three-dimensional orientation, amino acidcomposition and active site) and catalytic activity (rate and kineticsof cellobiose hydrolysis and ability to act on other substrates). Theβ-glucosidase enzyme may come from various sources; however, in allcases, the β-glucosidase enzyme can hydrolyze cellobiose to glucose. Theβ-glucosidase enzyme may be a Family 1 or Family 3 glycoside hydrolase,although other family members may be used in the practice of thisinvention. The preferred β-glucosidase enzyme for use in this inventionis the Bgl1 protein from Trichoderma reesei. Other forms might includeother Bgl proteins from Trichoderma or β-glucosidase enzymes from otherorganisms.

The binding of the β-glucosidase to the pretreated feedstock is effectedby a binding agent that binds the β-glucosidase enzyme to the pretreatedlignocellulosic feedstock. By the term “binding agent”, it is meant anychemical compound for binding the β-glucosidase to the fiber solids. Theaffinity of the binding agent for the pretreated feedstock is strongenough to allow the β-glucosidase enzyme to adhere to the fiber solidsin the aqueous feedstock slurry, thereby allowing it to be retained inthe hydrolysis reactor for a longer period of time than the aqueousphase of the slurry.

The binding agent may be a chemical attached to the β-glucosidase enzymein the form of a chemical modification. This modification involvesattaching to the enzyme a chemical with sufficient affinity for thefiber solids. Examples of suitable chemicals include detergents,surfactants, polyglycols, proteins and protein fragments. Examples ofdetergents and surfactants include, but are not limited to, bile acids(cholate, deoxycholate, taurocholate, glycocholate, andglycodeoxycholate are examples), alkyl glycosides(n-nonyl-β-D-glucopyranoside, n-octyl-β-D-glucopyranoside,n-heptyl-β-D-glucopyranoside, n-hexyl-β-D-glucopyranoside,dodecyl-β-D-maltoside octyl-β-D-thioglucopyranoside, glucopyranoside,and decyl-β-D-maltoside are examples) and zwittergents. Examples ofpolyglycols include, but are not limited to, polyethylene glycol andpolyoxyethylenes.

The binding agent may also be a protein or protein fragment. Examples ofproteins and protein fragments include those described above for use asbinding domains. Further examples of proteins that can serve as abinding agent include, but are not limited to, hydrophobin,streptolysin, swollenin or expansin. Examples of protein fragments thatcan serve as binding agents include, but are not limited to,polytryptophan, polytyrosin and amphipathic helices.

Preferably, the binding agent is a binding domain such as acarbohydrate-binding module (CBM) that is operably linked to theβ-glucosidase enzyme. By the term “carbohydrate-binding module” or“CBM,” it is meant any protein or peptide sequence that non-covalentlybinds to carbohydrate(s) present in the fiber solids. Preferably, thecarbohydrate-binding module is a cellulose-binding domain (CBD) thatbinds to cellulose in the fiber solids.

CBDs are found in nature as discrete domains in proteins such ascellulases and also in non-hydrolytic enzymes. To date, over twenty-fivefamilies of CBD sequences have been identified. The CBD for the practiceof this invention may be derived from any source of CBDs. For example,the CBD may be derived from a bacteria or fungus, although CBDs havebeen isolated from a variety of other organisms. Non-limiting examplesof microbes that the CBD may be derived from include Aspergillus,Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.Preferred CBD sequences for the practice of the invention are Type ICBDs, which are derived from fungi. Alternatively, the DNA sequenceencoding a CBD may be prepared synthetically by methods known to thoseof skill in the art such as the phosphoramidite method (Beaucage andCaruthers, Tetrahedron Letters, 1981, 22:1859-1869).

The term “operably linked” refers to a linkage between the β-glucosidaseenzyme and the binding domain which enables the binding domain to adhereto the fiber solids in the aqueous slurry. The linkage may be via alinker or the binding domain may be linked to the β-glucosidase withoutan intervening linker region.

A further example of a binding agent that may be used in the practice ofthe invention is a chemical that associates with both the β-glucosidaseenzyme and the fiber solids. Non-limiting examples of such chemicalsmight include, but are not limited to, polycations, polyanions,flocculents and amphipathic molecules. Furthermore, this chemical may bea protein or protein fragment, such as those described above for use asbinding domains, or a chemical such as those described above for use inchemical modification.

By the term “linker”, it is meant an amino acid sequence adjoining thecellulose-binding domain of a cellulase or β-glucosidase enzyme andconnecting it to the catalytically active domain of the enzyme. Thelinker region may be hydrophilic and uncharged and enriched in certainamino acids, including glycine, asparagine, proline, serine, threonine,glutamine, or combinations thereof. Preferably, the structure of thelinker imparts flexibility to the sequence. While not wishing to bebound by theory, the flexible structure is believed to facilitate theactivity of the catalytic domain. However, as would be evident to one ofskill in the art, it is not essential that a linker is present.

The ability of a β-glucosidase enzyme to bind to cellulose may bedetermined by cellulose-binding assays using pretreated lignocellulosicmaterial. Such assays are familiar to those skilled in the art andinvolve contacting 5 grams of pretreated lignocellulosic material with50 mg β-glucosidase enzyme, with binding agent, in an aqueous solutionfor 5 to 15 minutes at a temperature of 20° C. to 40° C., thenseparating the fiber solids from the enzyme by filtration and measuringthe amount of enzyme remaining in solution. The binding agent binds tothe β-glucosidase and the fiber solids, thereby allowing theβ-glucosidase enzyme to be retained in the hydrolysis reactor along withthe fiber solids.

Any source of β-glucosidase may be used in the practice of theinvention. For example, the β-glucosidase enzyme may be derived fromAspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or acombination thereof. Preferably, the β-glucosidase enzyme is derivedfrom Trichoderma or Aspergillus. The β-glucosidase enzyme derived fromTrichoderma is of molecular weight 74,000 (as measured bySDS-polyacrylamide gel electrophoresis) and has an isoelectric point of8.3 (as measured by non-denaturing isoelectric focusing polyacrylamidegel electrophoresis). The β-glucosidase enzyme may be native to thehost, or may be native to another genus or species and inserted into thehost to be expressed.

The β-glucosidase containing a CBM, such as a CBD, may be a fusionprotein produced by a genetic construct comprising a promoter sequence,a sequence encoding β-glucosidase and a sequence encoding a CBM. Thegenetic construct is expressed in a suitable expression system, forexample, a bacterial of fungal expression system such as Aspergillus,Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.In addition, naturally occurring β-glucosidase enzymes with a CBM may beused in the practice of the invention. Naturally occurring β-glucosidaseenzymes may be isolated from Aspergillus, Humicola, Trichoderma,Bacillus, Thermobifida, or a combination thereof. For example, anaturally occurring CBD-containing β-glucosidase has been purified andcharacterized from the white-rot fungus Phanaerochaete chrysosporium(Lymar et al., Appl. Environ. Micro., 1995, 61: 2976-2980, the contentsof which are incorporated herein by reference).

The dosage level of the β-glucosidase which is added to the aqueousslurry may be about 5 to about 400 β-glucosidase units per gram ofcellulose, or any amount therebetween, or from about 35 to about 200β-glucosidase units per gram of cellulose, or any amount therebetween.The β-glucosidase unit is measured according to the method of Ghose(supra).

It is preferred that the concentration of β-glucosidase present is highenough to ensure that cellobiose does not accumulate during thehydrolysis and inhibit the action of cellulase. It will be understood bythose of skill in the art that Trichoderma, and othercellulase-producing microbes, usually produce only limited amounts ofβ-glucosidase. The methods set forth in White and Hindle, U.S. Pat. No.6,015,703 (which is incorporated herein by reference) may be employed toachieve enhanced levels of production of β-glucosidase by Trichoderma.Alternately, β-glucosidase may be produced in a separate Aspergillusfermentation and added to the cellulase mixture.

It should be appreciated that not all of the β-glucosidase in the enzymecomposition may bind to the solids. For example, the amount ofβ-glucosidase enzyme present in the enzyme composition that comprises aCBD may be about 75% to about 100% (w/w), or any range therebetween, orabout 85% to about 100% (w/w), or any range therebetween, or about 90%to about 100% (w/w), or any range therebetween, of the totalβ-glucosidase present. For example, the amount of β-glucosidasecomprising a CBD in relation to the total amount of β-glucosidasepresent in the enzyme composition may be about 75, 78, 80, 83, 85, 87,90, 93, 95, 97, or 100% (w/w).

The cellulase enzymes and β-glucosidase enzymes may be handled in anaqueous solution, or as a powder or granulate. The enzymes may be addedto the aqueous slurry at any point prior to its introduction into ahydrolysis reactor. Alternatively, the enzymes may be added directly tothe hydrolysis reactor, although addition of enzymes prior to theirintroduction into the hydrolysis reactor is preferred for optimalmixing. The enzymes may be mixed into the aqueous slurry using mixingequipment that is familiar to those of skill in the art.

FIG. 1A is a non-limiting example of how the cellulase hydrolysis may becarried out on a lignocellulosic feedstock pretreated as describedabove. Prior to cellulase hydrolysis, the aqueous feedstock slurry 10 iscooled. This may be carried out by using a first heat exchanger 20 thatexchanges against glucose product stream 30 or other suitable fluid. Theaqueous slurry 10 may then be further cooled using a second fluid, forexample, cold water 45, at second heat exchanger 50. The slurry 10 maythen be pumped into a hydrolysis make-up tank 60, along with cellulaseenzymes and a β-glucosidase enzyme 70 having a cellulose-binding domain,and ammonia 80 to adjust the pH. In this example, the contents of thehydrolysis make-up tank 60 are mixed and pumped out of the make-up tank60, along pipe 120, to a hydrolysis tank 130. The make-up tank 60 may beused for adjusting the pH and achieving the desired temperature of theslurry.

It will be apparent to those of skill in the art that the enzymes may bemixed with the pretreated lignocellulosic feedstock slurry elsewhere,for example, within a line that feeds the make-up tank 60, including,but not limited to, upstream of first heat exchanger 20, a point betweenthe first 20 and second heat exchanger 50, or a point just prior toentry of the feedstock to the make-up tank 60. The enzymes may also beadded to the pretreated lignocellulosic feedstock slurry 10 after itexits the make-up tank 60; for example, they may be added to pipe 120.

By the term “hydrolysis reactor”, it is meant a reaction vessel used tocarry out partial or complete hydrolysis of the pretreatedlignocellulosic feedstock slurry by cellulase enzymes. The hydrolysisreactor must be of appropriate construction to accommodate thehydrolysis. A hydrolysis reactor may be jacketed with steam, hot water,or other heat source, to maintain the desired temperature. A hydrolysisreactor may be a tower with a height to diameter ratio of greater than2:1, or a tank with a height to diameter ratio of less than 2:1.

The term “solids-retaining hydrolysis reactor” as used herein refers toa hydrolysis reactor that retains fiber solids longer than the aqueousphase of the aqueous slurry to increase the reaction time of thecellulase and β-glucosidase enzymes with cellulose. The fiber solids areretained in the solids-retaining hydrolysis reactor by settling,filtration, centrifugation, or other means that partially or totallyseparate the aqueous phase from the fiber solids.

The hydrolysis may be carried out in a solids-retaining hydrolysisreactor that is part of a hydrolysis system that comprises one or morethan one hydrolysis reactor. The term “hydrolysis system” encompasseshydrolysis reactors as well as feed tanks, pumps, and other ancillaryequipment. The choice of the number of hydrolysis reactors in thehydrolysis system depends on the cost of hydrolysis reactors, the volumeof the aqueous slurry, and other factors. For a commercial-scale ethanolplant, the typical number of hydrolysis reactors is 4 to 12.

A solids-retaining hydrolysis reactor may be an unmixed hydrolysisreactor in the sense that no mechanical agitation of the reactorcontents is carried out during the hydrolysis reaction. An example of anunmixed hydrolysis reactor suitable for the practice of the invention isan upflow reactor which is described in co-pending U.S. patentapplication No. 60/637,189 (Foody et al.), which is incorporated hereinby reference. The solids-retaining hydrolysis reactor may also be amixed reactor, in which case mechanical agitation of the reactorcontents is carried out during the hydrolysis reaction. The activemixing within the hydrolysis tanks may be achieved by impellers or pumpsas is well known in the art.

If the solids-retaining hydrolysis reactor is a tower, it may be anupflow tower in which the aqueous slurry and enzymes enter the towerdirectly at the bottom of the tower and are pumped upward through thetower. Alternatively, the tower may be a downflow tower in which theaqueous slurry is pumped downward through the tower. The upflow ordownflow towers may be unmixed. Alternatively, there may be mixing atdiscreet levels.

Regardless of the number or type of hydrolysis reactors used, the fibersolids are retained in at least one solids-retaining hydrolysis reactorfor a longer time than the aqueous phase. Retention of the fiber solidsin the solids-retaining hydrolysis reactor can be accomplished byseveral means, which are discussed below. Regardless of how the fibersolids are retained in the solids-retaining hydrolysis reactor, thebinding of β-glucosidase to the fiber solids allows the β-glucosidase tobe retained with the fiber solids and not be withdrawn with the aqueousphase. This reduces the concentration of cellobiose relative to thatwhich would be present if β-glucosidase was withdrawn from thehydrolysis.

As the hydrolysis is carried out, the cellulose, lignin, and otherinsoluble components comprise the “fiber solids” of the aqueous slurry.The insoluble components, in addition to cellulose, that may be presentin the fiber solids include unconverted solids that are not digested bythe cellulase enzymes, as well as non-lignocellulosic components, orother compounds that are inert to cellulase, such as lignin and silicacompounds.

As the enzymatic hydrolysis continues the concentration of glucose inthe aqueous phase increases. In addition, low concentrations ofcellobiose may be present as well, although preferably in lowconcentrations. Additional soluble components that may be present in theaqueous phase include glucose oligomers, xylose and xylose oligomers,sugar degradation products such as furfural and hydroxyl methylfurfural, organic acids such as acetic acid, and phenolic compoundsderived from lignin. The term “aqueous phase” includes the aqueousportion of the enzyme hydrolyzed slurry and is not meant to be limitedto a separated aqueous phase of the slurry.

The hydrolysis may be carried out so that the total length of time thatthe aqueous phase resides in the solids-retaining hydrolysis reactor isabout 6 hours to about 200 hours. Preferably, the length of time of theaqueous phase in the hydrolysis is 12 hours to 96 hours. Morepreferably, the length of time of the aqueous phase in the hydrolysis is12 hours to 48 hours.

The fiber solids may be retained in the solids-retaining hydrolysisreactor for about 6 hours to about 148 hours longer than the aqueousphase, or any time therebetween, or between about 12 hours to about 148hours longer than the aqueous phase, or any time therebetween. Forexample, the fiber solids may be retained in the solids-retaininghydrolysis reactor for about 6, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, 100, 110, 120 or 148 hours longer than the aqueous phase.

Calculating the length of time the aqueous phase is in the hydrolysisreactor, or the residence time of the aqueous phase, depends on variousfactors. However, for the purpose of this specification, the length oftime the aqueous phase is in the hydrolysis reactor is determined simplyby dividing the working volume of the reactor by the volumetric slurryflow feed rate to the reactor. The length of time the unhydrolyzedsolids are retained in the hydrolysis reactor is determined based on thesolids concentration in the feed, exit and within the hydrolysisreactor, as is familiar to those of skill in the art.

The solids retention time in a hydrolysis reactor is calculated by (i)using a tracer compound that binds to the solids, or (ii) measuring theconcentration of lignin solids in the system. Method (i) is preferredbecause it is a more direct measure of the solids retention time.

An example of a tracer that binds to lignocellulosic solids is bovineserum albumin (BSA), but in principle any compound that binds to ligninand is detectable is suitable for this purpose. Using BSA as an exampletracer, it is added to the feed stream to the hydrolysis reactor at arate of 50-500 mg protein per gram solids, for 5 minutes, withoutotherwise disturbing the hydrolysis. Samples of 5 to 50 mL are thentaken every 10 minutes from the exit of the reactor. The samples arefiltered and washed with water. The BSA protein concentration on thesolids is measured by Kjeldahl nitrogen analysis, which is familiar tothose skilled in the art. The mean retention time of the solidscorresponds to the time at which 50% of the BSA has exited the reactor.

The second method to measure retention time is to take samples from atleast five locations throughout the reactor and measure theconcentration of lignin. The locations are best chosen at the range ofheights in the reactor vessel. The lignin concentration is measured bythe Klason lignin assay. The mean solids residence time is then theaverage lignin concentration divided by the lignin concentration of thefeed times the liquid retention time.

Referring again to FIG. 1A, in a non-limiting example, the hydrolyzedslurry comprising glucose and unhydrolyzed fiber solids is removed fromthe top of the solids-retaining hydrolysis reactor 130 via line 170 andintroduced to a microfiltration unit 180. The microfiltration unit 180separates the fiber solids comprising cellulose from the aqueous phaseof the hydrolyzed slurry. It should also be appreciated by those ofskill in the art that the fiber solids comprise entrapped liquor. Ifdesired, these separated fiber solids (line 195) are re-suspended in asecond hydrolysis reactor 200 and the hydrolysis is allowed to continue.

As described previously, during the hydrolysis, cellulases are bound tocellulose in the pretreated lignocellulosic feedstock. The β-glucosidaseenzyme, which binds to the pretreated lignocellulosic feedstock, willalso be bound to the fiber solids. Thus, when the fiber solids areseparated from the aqueous phase of the slurry, not only willexo-cellobiohydrolases (CBH) and endoglucanases (EG) remain with thefiber solids phase, but also β-glucosidase.

A number of methods could be employed to retain the fiber solids in thehydrolysis reactor for a longer time than the aqueous phase. These caninclude methods that almost completely separate the fiber solids fromthe aqueous phase, and methods that only partially separate the fibersolids from the aqueous phase. For example, the fiber solids may beseparated almost completely from the aqueous phase by membranefiltration, centrifugation, or vacuum or pressure filtration andreturned to the reactor. A preferred method of membrane filtration ismicrofiltration and a preferred method of centrifugation involvespumping the slurry through a hydroclone. These separation techniquesresult in a substantially complete separation of fiber solids particlesfrom the aqueous phase. The use of an upflow hydrolysis reactor, upflowsettler-clarifier, or hydrolysis reactors with low enough agitation toallow the fiber solids to settle would be suitable for the practice ofthis invention since these operations partially separate fiber solidsparticles from the aqueous phase and thereby retain the fiber solids inthe system for a longer time than the aqueous phase. In practicing theinvention with these systems, the β-glucosidase would remain bound tothe fiber solids and be retained in the hydrolysis. The unhydrolyzedfiber solids may be removed from the solids-retaining reactor togetherwith the aqueous phase or aqueous portion of the hydrolyzed slurry as asingle stream, or they may be removed as separate streams. If theaqueous portion is removed separately from the fiber solids, it shouldbe appreciated that separation may not be complete in that the streamcomprising the fiber solids may comprise a portion of the aqueous phaseand the aqueous phase may contain fiber solids.

A preferred method for carrying out the invention, which is not meant tobe limiting, involves carrying out the hydrolysis in a settling reactoras described in WO 2006/063467 (the contents of which are hereinincorporated by reference). An example of a hydrolysis systemincorporating upflow hydrolysis reactors is shown in FIG. 1B. Referencenumbers which are the same as in FIG. 1A indicate identical processsteps. As shown in FIG. 1B, the aqueous slurry is fed via line 120 tohydrolysis reactor 130. This can be by a line that goes down through themiddle of the reactor and then adds the slurry at the bottom, throughdistributor 140. Alternatively, the slurry feed can be directly todistributor 140 at the bottom of the reactor. The aqueous slurry flowsupward through the reactor with a vertical velocity that is low enoughto allow fiber solids to settle. As a result, the aqueous phasetraverses the reactor in a shorter time than the fiber solids. The boundcellulase and β-glucosidase remain in the reactor with the fiber solids.The bound β-glucosidase ensures that cellobiose is converted to glucosewithin the hydrolysis, and does not inhibit cellulase enzymes. Theunhydrolyzed solids are conveyed out of the reactor along with theaqueous phase at line 170 and are separated from the aqueous phase bymicrofiltration unit 180. The glucose stream is sent on to fermentationvia line 185, while the unhydrolyzed solids are conveyed (line 195) toline 160 for solids processing. (See FIG. 1A or 1B).

In an alternative method of carrying out the invention, the fiber solidsare retained in the hydrolysis reactor longer than the aqueous phase byseparating the fiber solids from the aqueous phase by microfiltrationand then returning the fiber solids to the reactor. Microfiltration isthe coarsest of the membrane filtration classes, which also includeultrafiltration and nanofiltration and is used to separate smallparticles suspended in liquids. The membranes used in microfiltrationare classified by pore diameter cutoff, which is the diameter of thesmallest particles that are retained by the membrane. The pore diametercutoff is typically in the range of about 0.1 to 10 microns.

The separated solids, obtained after a step of separating the fibersolids from the hydrolysis product comprising glucose may contain about50% to about 80% moisture. The moisture content depends on theseparation process used, the extent to which one chooses to de-water thesolids and the efficiency of water removal. The separated solids may bewashed with water to increase the amount of glucose removed.

After hydrolysis in a hydrolysis reactor with solids retention, thefiber solids may be separated, re-suspended and the hydrolysiscontinued. The fiber solids are resuspended in an aqueous phase which iscompatible for further hydrolysis of the re-suspended slurry. Theaqueous solution used for re-suspension of the solids is preferablywater, although other aqueous solutions may be used. The water may befresh water, process water, or steam condensate. The amount of aqueoussolution added for resuspension may be the same as was present in theaqueous slurry prior to hydrolysis, or preferably is somewhat less. Theminimum amount is that required to pump or convey and mix the slurry asneeded. The re-suspended slurry will be free of glucose and othersoluble inhibitors, or their concentrations significantly reduced. Inthe absence of glucose, cellobiose and inhibitors, or by decreasingtheir concentration, the step of further hydrolysis can be carried outwith increased efficiency.

Referring now to FIG. 1A, the re-suspension may be carried out byintroducing the separated solids via line 195 to a second hydrolysisreactor 200 along with water 210 and then re-suspending them to producea re-suspended slurry. The solids may be re-suspended in the liquid at asolids concentration of between about 3% and about 30% (w/w), or anyconcentration therebetween, for example, from about 10% to about 20%(w/w) suspended solids, or any concentration therebetween. Theconcentration of suspended solids in the re-suspended slurry ispreferably the same or somewhat higher than the concentration ofsuspended solids in the pretreated feedstock slurry prior to solidsseparation.

After the fiber solids are re-suspended, the hydrolysis is allowed tocontinue further to convert the cellulose to a product comprisingglucose. Hydrolysis of the re-suspended slurry may be allowed to proceedfor about another 24-120 hours; for example hydrolysis of there-suspended slurry may be allowed to proceed for about 12, 18, 24, 30,36, 42, 48, 54, 60, 66, 72, 90, or 120 hours. The bottom of the secondhydrolysis reactor 200 may be tapered to provide a path in which theheaviest solids may settle and be removed by pump 220 via line 230. (SeeFIG. 1B). These solids may then be sent for lignin processing 160.

Referring again to FIG. 1A, the hydrolyzed slurry, which comprisesglucose in the aqueous phase and unhydrolyzed solids and anyunhydrolyzed cellulose-containing particles in the fiber solids, may bewithdrawn from the top of the second hydrolysis reactor 200 via line 240and then introduced to a settling tank 250. The fiber solids settle tothe bottom of the settler tank 250. The aqueous phase 30 comprisingglucose may be removed via a pump. The unhydrolyzed solids may be pumpedout of the settler tank 250 via line 280.

The term “hydrolysis product” refers to products produced during theenzymatic hydrolysis, including, but not limited to glucose that ispresent in the aqueous phase. In addition to glucose, the aqueous phaseof the hydrolysis product may also comprise cellobiose, glucoseoligomers, or a combination thereof. Small amounts of unconvertedcellulose, as well as non-cellulosic materials, or other materials thatare inert to cellulase, may be carried over into the aqueous phase.These solids may be separated from the glucose stream to produce apreparation that is free of solid particles.

Although the system described above employs two hydrolysis reactors, theprocesses of hydrolysis with fiber solids retention, with or withoutcontinued hydrolysis of a re-suspended slurry, may be performed in asingle or in more than two hydrolysis reactors.

It should also be appreciated that, after the optional step of furtherhydrolysis, the re-suspended slurry may be subjected to furtherhydrolysis, separation of the solids phase from the aqueous phase andre-suspension of the separated solids to produce a re-suspended slurry.These steps may be repeated 1 to 5 times, or any number of timestherebetween, preferably 1 to 2 times.

Furthermore, the separated solids may be sent to one or more than oneupstream or downstream hydrolysis reactor throughout the processingsteps. For example, a first portion of the separated solids may berecycled to an upstream reactor and a second portion of the separatedsolids may be added to a downstream reactor.

The separated solids, obtained after a step of separating the fibersolids from the hydrolysis product comprising glucose may then be addedto a second aqueous solution comprising glucose obtained from anotherpart of the hydrolysis process to produce a combined sugar stream. Forexample, with reference to FIG. 1A, the aqueous solution containingglucose may be removed via line 195 and added to glucose stream 30.Alternatively, fermentation or further processing is carried outseparately on the aqueous phase produced during the initial hydrolysisand the re-suspended hydrolysis.

The glucose produced by the hydrolysis of cellulose from the pretreatedlignocellulosic feedstock may be fermented to ethanol. Fermentation ofglucose and other sugars to ethanol may be performed by conventionalprocesses known to those skilled in the art and may be effected by avariety of microorganisms including yeast and bacteria or geneticallymodified microorganisms, for example, but not limited to those describedin WO 95/13362, WO 97/42307, or as described in Alcohol Production FromLignocellulosic Biomass: The Iogen Process (in: The Alcohol Textbook,Nottingham University Press, 2000), which are herein incorporated byreference.

The present invention will be further illustrated in the followingexamples. However, it is to be understood that these examples are forillustrative purposes only, and should not be used to limit the scope ofthe present invention in any manner.

EXAMPLES Example 1 Hydrolysis of Pretreated Feedstock with CellulaseEnzymes and β-glucosidase Containing a CBD in an Upflow HydrolysisReactor

With reference to FIG. 1B, the pretreated feedstock slurry is preparedfrom 91 t/hr of wheat straw at 20% moisture. The straw is ground to 20mesh with a hammer mill and cooked with steam at 230° C. and 3314 kg/hrsulfuric acid (93% w/w) diluted in 422,000 kg/hr of water in accordancewith the teaching of Foody, U.S. Pat. No. 4,461,648. When exiting thepretreatment reactor, the pretreated lignocellulosic feedstock slurry 10is cooled using a heat exchanger 20 that exchanges against an aqueousglucose stream 30 or other suitable fluid. The pretreated feedstockslurry 10 is then further cooled to a temperature of between about 45°C. and about 55° C. using a second fluid, for example, cold water 45 atheat exchanger 50. The pretreated feedstock slurry 10 is then pumpedinto a hydrolysis make-up tank 60, along with an aqueous solution ofenzymes 70, which include cellulase enzymes from the fungus Trichodermaat a dosage of 19 IU per gram cellulose and a β-glucosidase enzymecomprising a CBD, made as described in Example 5, at a dosage of 145IU/g cellulose. This is the feed to the hydrolysis tower. However, itshould be noted that the enzymes 70 may also be added elsewhere; forexample, the enzymes 70 may be added within any line that feeds thehydrolysis reactor. Ammonia 80 is also added to the slurry 10 at a rateof 1463 kg/hr immediately prior to enzyme addition to adjust the pH tobetween about 4.5 and 5.0. The contents of the hydrolysis make-up tank60 are mixed with an agitator 100 and the slurry 10 is then is pumpedout of the make-up tank 60 by first pump 110, along pipe 120, to one ofseven similar hydrolysis reactors, of which hydrolysis reactor 130 isone such reactor operated in parallel trains.

The hydrolysis reactor 130 comprises distributors 140 for maintaining auniform distribution of the enzyme-treated slurry. The hydrolysisreactor 130 is an unmixed upflow settling reactor as described inco-pending U.S. patent application No. 60/637,189. The reactor is a tankof diameter 60 feet and height 60 feet. The slurry 10 is added to thebottom of the hydrolysis reactor 130 at a rate of 300 gpm and a fibersolids concentration of about 10% (w/w). The tank is tapered to providea path in which the heaviest solids settle and are removed by pump 142via line 145. These solids may be sent for lignin processing via line160 or recovered separately or discharged. The aqueous phase and fibersolids flow up the tank with the fiber solids settling and ascending thetank at a slower rate than the liquid.

The slurry exits the tank after a residence time of the aqueous phase ofabout 72 hours and of the fiber solids, which maintain a concentrationof 12% (w/w), of about 130 hours. The cellulose conversion is about 95%.The hydrolyzed slurry, which comprises an aqueous phase of 60 g/Lglucose and fiber solids comprising primarily unhydrolyzed cellulose aswell as lignin and silica, is removed from the top of the hydrolysisreactor 130 via line 170 and introduced to a microfiltration unit 180 ata rate of 300 gpm. The microfiltration unit 180 separates the fibersolids comprising cellulose, lignin and bound cellulase andβ-glucosidase from the aqueous phase. The aqueous phase contains littleenzyme with the glucose stream and is removed via line 185 and sent tofermentation to ethanol by yeast. The separated fiber solids containingbound cellulase and β-glucosidase in line 195 are combined with theheavy solids in line 160 and sent for solids processing.

Example 2 Hydrolysis of Pretreated Feedstock with Cellulase Enzymes andβ-glucosidase Containing a CBD in an Upflow Hydrolysis Reactor withContinued Hydrolysis

This example relates to the enzymatic hydrolysis of a pretreatedfeedstock with cellulase enzymes and β-glucosidase with a CBD, followedby separation of unhydrolyzed fiber solids from the aqueous phase andresuspension of the fiber solids. The re-suspended fiber solids, whichcontain the bound β-glucosidase enzyme and cellulase enzymes, arehydrolyzed in a second hydrolysis reactor.

Hydrolysis of pretreated feedstock with cellulase enzymes andβ-glucosidase enzyme with a CBD is carried out in an upflow hydrolysisreactor as described in Example 1. However, in this case, the dimensionsof the hydrolysis reactor are selected so that the liquid exits the tankafter a residence time of about 24 hours with a cellulose conversion ofabout 55% to produce a partially-hydrolyzed slurry 150. Thepartially-hydrolyzed slurry 150, which comprises an aqueous phase of 30g/L glucose and fiber solids comprising primarily unhydrolyzedcellulose, as well as lignin and silica, is removed from the top of thefirst hydrolysis reactor 130 via line 170 and introduced to amicrofiltration unit 180 at a rate of 900 gpm.

The microfiltration unit 180 separates the solids comprising cellulose,lignin, bound cellulase and β-glucosidase from the aqueous phase of thepartially-hydrolyzed slurry. The aqueous phase contains little enzymewith the glucose stream and is removed via line 185 and added to glucosestream 30. The separated solids in line 195 containing bound cellulaseand β-glucosidase are introduced to a second hydrolysis reactor 200along with water 210 to produce a re-suspended slurry and then fed tothe second hydrolysis reactor 200 which is also an upflow hydrolysisreactor. The feed rate to the second reactor is about 450 gpm and theliquid residence time is about 48 hours. Similar to the first hydrolysisreactor 130, the bottom of the second hydrolysis reactor 200 is taperedto provide a path in which the heaviest solids settle and are removed bypump 220 via line 230. These solids may then be sent for ligninprocessing via line 160 or removed separately or discharged.

Glucose, and any unhydrolyzed cellulose-containing and lignin-containingparticles are then withdrawn from the top of the second hydrolysisreactor 200 via line 240 and are introduced to a settling tank 250. Thesolids settle in the bottom of the settler tank 250 and the hydrolysisproduct stream 30 comprising glucose is removed via pump 260. Thesettled solids are pumped out of the settler tank 250 by pump 270 vialine 280. These solids are then sent for lignin processing 160. Stream30 is sent to the first heat exchanger or for fermentation to ethanol byyeast.

Example 3 Cellulose Hydrolysis by Enzyme Including β-glucosidase withCellulose Binding Domain (CBD)

This example illustrates the hydrolysis of pretreated cellulose withsolids separation and resuspension of the substrate. The performance ofthe hydrolysis is better with β-glucosidase with a CBD present thanwithout a CBD.

Pretreated wheat straw was prepared by continuous pretreatment with 0.6%sulfuric acid (w/w) on feedstock, heated to 185° C. with steam for 3minutes. The pretreated feedstock was washed with an excess of water andvacuum filtered to remove most of the water. The washed feedstock cakecontained 30% solids, and the solids contained 51% cellulose, with thebalance being composed primarily xylan, lignin and silica.

Two cellulase enzyme mixtures from Trichoderma submerged culturefermentations were used in this experiment. Both mixtures containedenhanced levels of β-glucosidase to ensure cellobiose did not accumulateduring the hydrolysis. The level of β-glucosidase was enhanced by themethods of White and Hindle, U.S. Pat. No. 6,015,703. One mixturecontained 163 g/L protein and 131 IU/mL filter paper cellulase activity.This batch (“conventional”) contained native β-glucosidase lacking acellulose binding domain. The β-glucosidase activity was measured by thestandard assay of Ghose (1987) as 1235 IU/mL. A second batch (“βg withCBD”) contained 32.5 g/L protein, 20.7 IU/mL filter paper cellulaseactivity, and 250 IU/mL β-glucosidase activity. Example 5 describes thepreparation of β-glucosidase with CBD in more detail.

Cellulose hydrolyses were carried out by using 250 mL screw top flasks.The total hydrolysis weight was 100 g per flask, with pretreated wheatstraw at a concentration corresponding to 5% cellulose, enzyme added ata dosage of 16 or 24 mg protein per gram of cellulose, and the balancecontaining 50 mM sodium citrate buffer, pH 4.8, which contained 0.5%sodium benzoate as a preservative. Before adding the enzyme, thepretreated wheat straw substrate was hydrated overnight in the buffer at50° C. with the flasks shaking. During the hydrolysis, the flasks wereshaken at 250 rpm in a 50° C. gyratory shaker.

For hydrolyses with filtration and resuspension, the flasks were removedfrom the shaker at 24 hours and the contents vacuum-filtered over glassmicrofiber filter paper. The filtrate volume was measured as 40-50 mLand the filtrate was replaced by an equal volume of 50 mM sodium citratebuffer, pH 4.8. Similar to the hydrolysis carried out prior tofiltration and resuspension, the shaken hydrolysis was then continuedfor 96 hours. For conventional hydrolyses, the hydrolysis runs werecarried out shaken for 120 hours without filtration or resuspension.

For all hydrolyses, 800 μL samples were periodically taken andtransferred into micro-centrifuge filters and centrifuged at 12,000 rpmfor 2 minutes to separate the insoluble solids from the aqueous phase.The supernatant was recovered and used for glucose analysis. Mostsamples were checked to ensure cellobiose did not accumulate by boilingfor 5 minutes prior to centrifugation.

Glucose concentrations in the supernatant were measured by an enzymaticmethod. Low (<1 g/L) cellobiose concentrations were confirmed bymeasurement on an HPLC. A cellulose assay based on hydrolysis withconcentrated sulfuric acid was performed at the end of all hydrolysisruns and confirmed the concentration of unconverted cellulose based onglucose measurement.

FIG. 2A shows the results of hydrolysis by cellulase with β-glucosidasecontaining CBD, with cellulase dosages of 16 mg protein per gramcellulose. The re-suspended hydrolysis outperforms the conventionalhydrolysis that was carried out without resuspension. The reason is thatthe filtration of the hydrolysis after 24 hours removes a significantamount of the glucose present. By removing the glucose, the end productinhibition of the cellulase is removed, and the hydrolysis proceeds at ahigher rate and reaches a higher level of conversion than in thepresence of glucose in the conventional hydrolysis. The β-glucosidase,which is necessary for an effective hydrolysis, is bound to thecellulose and is carried into the resuspension hydrolysis.

FIG. 2B shows a similar result as FIG. 2A, except the enzyme dosage is24 mg/g instead of 16 mg/g in FIG. 2A.

FIG. 3 shows hydrolysis with a conventional cellulase, where theβ-glucosidase lacks a CBD. The hydrolyses were carried out for 24 hoursat dosages of 16 and 24 mg/g. At this point, the slurries were filteredand the hydrolyses re-suspended and continued. The rate of hydrolysisafter re-suspension is very low, with very little glucose produced. Thereason for this low rate of hydrolysis is that the β-glucosidase lacks aCBD and does not bind to the cellulose, but rather is lost to thefiltrate during filtration. The buildup of cellobiose inhibits thecellulase and slows down the rate of hydrolysis.

Example 4 Binding of β-glucosidase with CBD to Bleached Wheat StrawCellulose

β-glucosidase and β-glucosidase containing a CBD were purified fromwhole cellulase mixtures by anion exchange chromatography followed bycation exchange chromatography. The purified proteins were incubatedwith 2.56 g/L pretreated wheat straw adjusted to pH 4.8 with citratebuffer or with pH 4.8 citrate buffer alone for 30 minutes at 4° C. or50° C. Following incubation, the samples were centrifuged and thesupernatant fractions were analyzed by SDS-PAGE (FIGS. 4A and 4B).

As shown in FIG. 4A, after incubation at 4° C. in the presence andabsence of pretreated wheat straw, identical amounts of β-glucosidasewere detected in the supernatant. This is indicated by the bands at 66kDa and indicates that β-glucosidase lacking a CBD did not bind to thepretreated wheat straw. In contrast, purified β-glucosidase-CBDcompletely bound to pretreated wheat straw and was not detected in thesupernatant, as indicated by the band at 70 kDa in the absence ofpretreated wheat straw, and the absence of the band in the presence ofpretreated wheat straw. This shows that the CBD is required forβ-glucosidase to bind to the fiber solids. Similar results were observedat 50° C. (FIG. 4B).

Example 5 Expression of a β-glucosidase/CBD Fusion in Trichoderma reesei

This example describes the isolation of genomic DNA from Trichodermareesei strain M2C38 and genetically modified derivatives, theconstruction of genomic DNA libraries, the cloning of various genes,genetic constructs from Trichoderma reesei strain M2C38, and thetransformation and expression of β-glucosidase/CBD genetic constructs inTrichoderma reesei strain BTR213.

Trichoderma reesei strains M2C38 and BTR213 are proprietary strains ofIogen Corporation which were derived from Trichoderma reesei RutC30(ATCC 56765, Montenecourt and Eveleigh, Adv. Chem. Ser., 1979, 181:289-301), which was, in turn, derived from Trichoderma reesei Qm6A (ATCC13631 Mandels and Reese, J. Bacteriol., 1957, 73: 269-278).

In this example, restriction endonucleases, T4 DNA polymerase, T4 DNAligase and Klenow fragment of E. coli DNA polymerase 1 were purchasedfrom Gibco/BRL, New England Biolabs, Boehringer Mannheim or Pharmaciaand used as recommended by the manufacturer. Pwo polymerase withproof-reading activity (Boehringer Mannheim) was used in allpolymerase-chain reactions (PCR) according to the manufacturer'sprotocol. Hygromycin B was purchased from CalBiochem.

5.1 Cloning of the T. reesei bgl1, cbh1, cbh2, xln2 and pgk Genes.

To isolate genomic DNA, 50 mL of Potato Dextrose Broth (Difco) wasinoculated with T. reesei spores collected from a Potato Dextrose Agarplate with a sterile inoculation loop. The cultures were shaken at 200rpm for 2-3 days at 28° C. The mycelia were filtered onto a GFA glassmicrofibre filter (Whatman) and washed with cold deionized water. Thefungal cakes were frozen in liquid nitrogen crushed into a powder with apre-chilled mortar and pestle; 0.5 g of powdered biomass werere-suspended in 5 mL of 100 mM Tris, 50 mM EDTA, pH 7.5 plus 1% sodiumdodecyl sulphate (SDS). The lysate was centrifuged (5000 g for 20 min,4° C.) to pellet cell debris. The supernatant was extracted with 1volume buffer-(10 mM Tris, 1 mM EDTA, pH 8.0)-saturated phenol, followedby extraction with 1 volume of buffer-saturatedphenol:chloroform:isoamyl alcohol (25:24:1) in order to remove solubleproteins. DNA was precipitated from the solution by adding 0.1 volumesof 3 M sodium acetate, pH 5.2 and 2.5 volumes of cold 95% ethanol. Afterincubating for at least 1 hour at −20° C., the DNA was pelleted bycentrifugation (5000 g for 20 min, 4° C.), rinsed with 10 mL 70%ethanol, air-dried and re-suspended in 1 mL 10 mM Tris, 1 mM EDTA, pH8.0. RNA was digested by the addition of Ribonuclease A (BoehringerMannheim) added to a final concentration of 0.1 mg/mL and incubated at37° C. for 1 hour. Sequential extractions with 1 volume ofbuffer-saturated phenol and 1 volume of buffer-saturatedphenol:chloroform:isoamyl alcohol (25:24:1) were used to remove theribonuclease from the DNA solution. The DNA was again precipitated with0.1 volumes of 3 M sodium acetate, pH 5.2 and 2.5 volumes of cold 95%ethanol, pelleted by centrifugation, rinsed with 70% ethanol, air-driedand re-suspended in 50 μl of 10 mM Tris, 1 mM EDTA, pH 8.0. Theconcentration of DNA was determined by measuring the absorbance of thesolution at 260 nm (p. C1 in Sambrook, Fritsch and Maniatis, “MolecularCloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Press1989, hereafter referred to as Sambrook et al.).

Two plasmid libraries and one phage library were constructed usinggenomic DNA isolated from T. reesei strain M2C38. The plasmid librarieswere constructed in the vector pUC119 (Viera and Messing, “Isolation ofsingle-stranded plasmid DNA”, Methods Enzymol. 153:3, 1987) as follows:10 μg genomic DNA was digested for 20 hrs at 37° C. in a 100 μL volumewith 2 units/μg of HindIII, BamH1 or EcoR1 restriction enzymes. Thedigested DNA was fractionated on a 0.75% agarose gel run in 0.04MTris-acetate, 1 mM EDTA and stained with ethidium bromide. Gel slicescorresponding to the sizes of the genes of interest (based on publishedinformation and Southern blots) were excised and subjected toelectro-elution to recover the DNA fragments (Sambrook et al., pp.6.28-6.29). These enriched fractions of DNA were ligated into pUC119 inorder to create gene libraries in ligation reactions containing 20-50μg/mL DNA in a 2:1 molar ratio of vector:insert DNA, 1 mM ATP and 5units T4 DNA ligase in a total volume of 10-15 μl at 4° C. for 16 h.Escherichia coli strain HB101 was electroporated with the ligationreactions using the Cell Porator System (Gibco/BRL) following themanufacturer's protocol and transformants selected on LB agar containing70 μg/mL ampicillin.

E. coli HB101 transformants harboring cbh1, cbh2 or bgl1 clones from therecombinant pUC119-Hind III, -BamH1 or -EcoR1 libraries were identifiedby colony lift hybridization: 1-3×10⁴ colonies were transferred ontoHyBond™ nylon membranes (Amersham); membranes were placed colony-side uponto blotting paper (VWR 238) saturated with 0.5 M NaOH, 1 M NaCl for 5minutes to lyse the bacterial cells and denature the DNA; the membraneswere then neutralized by placing them colony-side up onto blotting paper(VWR 238) saturated with 1.5 M Tris, pH 7.5 plus 1 M NaCl for 5 min; themembranes were allowed to air-dry for 30 min and the DNA was then fixedto the membranes by baking at 80° C. for 2 h.

³²P-labelled probes were prepared by PCR amplification of short (0.7-1.5kB) fragments of the bgl1, cbh1 and cbh2 coding regions from theenriched pool of HindIII, BamH1 or EcoR1 fragments, respectively, in alabelling reaction containing 10-50 ng target DNA, 0.2 mM each d(GCT)TP,0.5 μM dATP, 20-40 μCi α-³²P-dATP, 10 pmole oligonucleotide primers and0.5 units Taq polymerase in a total volume of 20 μL. The reaction wassubjected to 6-7 cycles of amplification (95° C., 2 min; 56° C., 1.5min; 70° C., 5 min). The amplified, ³²P-labelled DNA was precipitated bythe addition of 0.5 mL 10% (w/v) trichloroacetic acid and 0.5 mg yeasttRNA. The DNA was pelleted by microcentrifugation, washed twice with 1mL 70% ethanol, air-dried and re-suspended in 1M Tris pH7.5, 1 mM EDTA.

Nylon membranes onto which the recombinant pUC119 plasmids had beenfixed were prehybridized in heat-sealed bags for 1 h at 60-65° C. in 1 MNaCl, 1% SDS, 50 mM Tris, 1 mM EDTA pH 7.5 with 100 μg/mL denaturedsheared salmon sperm DNA. Hybridizations were performed in heat-sealedbags in the same buffer with only 50 μg/mL denatured sheared salmonsperm DNA and 5×10⁶-5×10⁷ cpm of denatured bgl1, cbh1 or cbh2 probe for16-20 h at 60-65° C. Membranes were washed once for 15 minutes with 1 MNaCl, 0.5% SDS at 60° C., twice for 15 minutes each with 0.3M NaCl, 0.5%SDS at 60° C. and once for 15 minutes with 0.03M NaCl, 0.5% SDS at 55°C. Membranes were again placed in heat-sealed bags and exposed to KodakRP X-ray film to 16-48 h at −70° C. The X-ray film was developedfollowing the manufacturer's protocols. Colonies giving strong or weaksignals were picked and cultured in 2xYT media supplemented with 70μg/mL ampicillin. Plasmid DNA was isolated from these cultures using thealkaline lysis method (Sambrook, et al., pp. 1.25-1.28) and analyzed byrestriction digest, Southern hybridization (Sambrook, et al., pp.9.38-9.44) and PCR analysis (Sambrook, et al., pp. 14.18-14, 19).

Clones carrying the bgl1 gene were identified by colony lifthybridization of the pUC119-Hind III library with a 1.0 kb bgl1 probeprepared using oligonucleotide primers designed to amplify bp 462-1403of the published bgl1 sequence (Barnett, Berka, and Fowler, in “Cloningand Amplification of the Gene Encoding an Extracellular β-glucosidasefrom Trichoderma reesei: Evidence for Improved Rates of Saccharificationof Cellulosic Substrates” Bio/Technology, Volume 9, June 1991, p.562-567, herein referred to as “Barnett, et al.”). A bgl1 clone,pJEN200, was isolated containing 6.0 kb Hind III fragment correspondingto the promoter, structural gene and termination sequences. Clonescarrying the cbh1 gene were identified by colony lift hybridization ofthe pUC119-BamH1 library with a 0.7 kb cbh1 probe prepared usingoligonucleotide primers designed to amplify bp 597-1361 of the publishedcbh1 sequence (Shoemaker, Schweikart, Ladner, Gelfand, Kwok, Myambo andInnis, “Molecular cloning of exo-cellobiohydrolyase 1 derived fromTrichoderma reesei strain L27”, Bio/Technology 1: 691-696, 1983hereafter referred to as Shoemaker et al.). A cbh1 clone, pCOR132 wasisolated containing a 5.7 kb BamH1 fragment corresponding to thepromoter (4.7 kb) and 1 kb of the cbh1 structural gene. From this, a 2.5kb EcoR1 fragment containing the cbh1 promoter (2.1 kb) and 5′ end ofthe cbh1 coding region (0.4 kb) was subcloned into pUC119 to generatepCB152. Clones carrying the cbh2 gene were identified by colony lifthybridization of the pUC119-EcoR1 library with a 1.5 kb cbh2 probeprepared using oligonucleotide primers designed to amplify bp 580-2114of the published cbh2 sequence (Chen, Gritzali and Stafford, “Nucleotidesequence and deduced primary structure of cellobiohydrolase II fromTrichoderma reesei”, Bio/Technology 5: 274-278, 1987, hereafter referredto as Chen et al.). A cbh2 clone, pZUK600 was isolated containing a 4.8kb EcoR1 fragment corresponding to the promoter (600 bp), structuralgene (2.3 kb) and terminator (1.9 kbp).

A phage library was constructed in the lambda vector λDASH (Stratagene,Inc.) as follows: genomic DNA (3 μg) was digested with 2, 1, 0.5 and 0.5units/μg Bam HI for 1 hour at 37° C. to generate fragments 9-23 kB insize. The DNA from each digest was purified by extraction with 1 volumeTris-saturated phenol:choroform:isoamyl alcohol (25:24:1) followed byprecipitation with 10 μl 3M sodium acetate, pH 5.2 and 250 μl 95%ethanol (−20° C.). The digested DNA was pelleted by microcentrifugation,rinsed with 0.5 mL cold 70% ethanol, air-dried and re-suspended in 10 μLsterile, deionized water. Enrichment of DNA fragments 9-23 kB in sizewas confirmed by agarose gel electrophoresis (0.8% agarose in 0.04 MTris-acetate, 1 mM EDTA). Digested DNA (0.4 μg) was ligated to 1 μgλDASH arms predigested with BamHI (Stratagene) in a reaction containing2 units T4 DNA ligase and 1 mM ATP in a total volume of 5 μL at 4° C.overnight. The ligation mix was packaged into phage particles using theGigaPack® II Gold packaging extracts (Stratagene) following themanufacturer's protocol. The library was titred using the E. coli hoststrain XL1-Blue MRA (P2) and found to contain 3×10⁵ independent clones.

Digoxigen-11-dUTP labelled probes were prepared from PCR amplifiedcoding regions of the cbh1, xln2 and pgk genes by random prime labellingusing the DIG Labelling and Detection kit (Boehringer Mannheim) andfollowing the manufacturer's protocols. Genomic clones containing thecbh1, xln2 and pgk genes were identified by plaque-lift hybridization ofthe λDASH library. For each gene of interest, 1×10⁴ clones weretransferred to Nytran® (Schleicher and Schull) nylon membranes. Thephage particles were lysed and the phage DNA denatured by placing themembranes plaque-side up on blotting paper (VWR238) saturated with 0.5 MNaOH, 1 M NaCl for 5 minutes; the membranes were then neutralized byplacing them plaque-side up onto blotting paper (VWR238) saturated with1.5 M Tris, pH 7.5 plus 1 M NaCl for 5 min; the membranes were allowedto air-dry for 30 min and the DNA was then fixed to the membranes bybaking at 80° C. for 2 hours. The membranes were prehybridized inheat-sealed bags in a solution of 6×SSPE, 5×Denhardt's, 1% SDS plus 100μg/mL denatured, sheared salmon sperm DNA at 65° C. for 2 h. Themembranes were then hybrized in heat-sealed bags in the same solutioncontaining 50 μg/mL denatured, sheared salmon sperm DNA and 0.5 μg ofdigoxigen-dUTP labelled probes at 65° C. overnight. The membranes werewashed twice for 15 min in 2×SSPE, 0.1% SDS at RT, twice for 15 minutesin 0.2×SSPE, 0.1% SDS at 65° C. and once for 5 minutes in 2×SSPE.Positively hybridizing clones were identified by reaction with ananti-digoxigenin/alkaline phosphatase antibody conjugate,5-bromo-4-chloro-3-indoyl phosphate and 4-nitro blue tetrazoliumchloride (Boehringer Mannheim) following the manufacturer's protocol.Positively hybridizing clones were purified further by a second round ofscreening with the digoxigen-dUTP labelled probes. Individual cloneswere isolated and the phage DNA purified as described in Sambrook et al.(1989) pp. 2.118-2.121, with the exception that the CsCl gradient stepwas replaced by extraction with 1 volume of phenol:choroform:isoamylalcohol (25:24:1) and 1 volume of chloroform:isoamyl alcohol (24:1). TheDNA was precipitated with 0.1 volumes of 3M sodium acetate, pH 5.2 and2.5 volumes cold 95% ethanol. The precipitated phage DNA was washed with0.5 μL cold 70% ethanol, air-dried and re-suspended in 50 μL 10 mM Tris,1 mM EDTA pH8.0. Restriction fragments containing the genes of interestwere identified by restriction digests of the purified phage DNA andSouthern blot hybridization (Sambrook, et al., pp. 9.38-9.44) using thesame digoxigen-dUTP labelled probes used to screen the λDASH library.The membranes were hybridized and positively hybridizing fragmentsvisualized by the same methods used for the plaque lifts. Once thedesired restriction fragments from each λDASH clone were identified, therestriction digests were repeated, the fragments were resolved on a 0.8%agarose gel in TAE and the desired bands excised. The DNA was elutedfrom the gel slices using the Sephaglas B and Prep Kit (Pharmacia)following the manufacturer's protocol.

Clones carrying the cbh1 gene were identified by colony lifthybridization of the λDASH library (example 2) with a cbh1 probecomprising bp 45-2220 of the published cbh1 sequence (Shoemaker et al.).A 1.8 kb BamHI fragment containing the 3′ end of the cbh1 coding region(0.5 kb) and the cbh1 terminator (1.3 kb) was isolated by restrictiondigestion of phage DNA purified from a λDASH cbh1 clone. This fragmentwas subcloned into the BamH1 site of the E. coli plasmid vector pUC119to generate the plasmid pCB1Ta. Clones carrying the xln2 gene wereidentified by colony lift hybridization of the λDASH library (example 2)with a xln2 probe comprising bp 100-783 of the published xln2 sequence(Saarelainen, Paloheimo, Fagerstrom, Suominen and Nevalainen, “Cloning,sequencing and enhanced expression of the Trichoderma reeseiendoxylanase II (pI 9) gene xln2”, Mol. Gen. Genet. 241: 497-503, 1993,hereafter referred to as Saarelainen et al.). A 5.7 kb Kpn1 fragmentcontaining the promoter (2.3 kb), coding region (0.8 kb) and terminator(2.6 kb) the xln2 gene was isolated by restriction digestion of phageDNA purified from a λDASH xln2 clone. This fragment was subcloned intothe Kpn1 site of pUC119 to generate the plasmid pXYN2K-2. Clonescarrying the pgk gene were identified by colony lift hybridization ofthe λDASH library (example 2) with a pgk1 probe comprising bp 4-1586 thepublished pgk sequence (Vanhanen, Penttila, Lehtovaara and Knowles,“Isolation and characterization of the 3-phosphoglycerate kinase gene(pgk) from the filamentous fungus Trichoderma reesei”, Curr. Genet. 15:181-186, 1989). A 5.0 kb EcoR1 fragment containing the promoter (2.9kb), coding region (1.6 kb) and terminator (0.5 kb) the pgk gene wasisolated by restriction digestion of phage DNA purified from a λDASH pgkclone. This fragment was subcloned into the EcoR1 site of pUC119 togenerate the plasmid pGK5.0.

5.2 Construction of β-glucosidase Overexpression Vector pC/XBG-CBD-TV

This Example describes the construction of a vector designed to expressa fusion protein of the mature β-glucosidase coding region and a peptidecomprising the linker-cellulose binding domain of Trichodermacellobiohydrolase I. In this construct, the expression of the fusionprotein is directed by the Trichoderma cellobiohydrolase I (cbh1)promoter and xylanase 2 (xln2) secretion signal peptide.

The β-glucosidase coding region less the C-terminal alanine residue (bp474-2679) was amplified with Pwo polymerase from the genomic bgl1 clonepJEN200 using primers to insert an Xba1 site directly upstream of bp 474in the published bgl1 sequence (Barnett et al.) and a Kpn1 site at bp2676, which is one codon away from the stop codon. This amplifiedfragment was subcloned without digestion into the Sma1 site of pUC19 togenerate the plasmid pBgns1. The bgl1 fragment lacking the stop codonwas released from pBgns1 by digestion with Xba1 and Kpn1 and insertedinto pCB219N digested with XbaI and Kpn1 to generate pBgns2. To makepCB219N, a cbh2 terminator fragment was amplified from the pZUK600template using a primer homologous to bp 2226-2242 of the published 3′untranslated region of the cbh2 gene (Chen et al., 1987) containing aKpn1 site at the 5′ end and the pUC forward primer (Cat. No. 1224, NewEngland Biolabs) which anneals downstream of the EcoR1 site at the 3′end of cbh2 in pZUK600. This fragment was digested at the engineeredKpn1 and EcoR1 sites and inserted into the corresponding sites of pUC119to generate pCB219. An EcoR1-Not1 adaptor (Cat. No. 35310-010,Gibco/BRL) was inserted into the unique EcoR1 site of pCB219 to generatepCB219N.

A 2.3 kb fragment containing the promoter and secretion signal of thexln2 gene (bp −2150 to +99 where +1 indicates the ATG start codon) wasamplified with Pwo polymerase from the genomic xln2 subclone pXYN2K-2using a xln2-specific primer containing a Nhe1 site directly downstreamof bp102 of the published xln2 sequence (Saarelainen et al.) and the pUCreverse primer (Cat. No. 18432-013, Gibco/BRL) which anneals upstream ofthe Kpn1 site at the 5′ end of the xln2 gene. This xln2 PCR product wasdigested with EcoR1 (which was amplified as part of the pUC119polylinker from pXYN2K-2) and Nhe1 and inserted into the plasmidpBR322L, which was prepared from the plasmid pBR322 by insertion of anSph1-Not1-Sal1 linker between the Sph1 and Sal1 sites. The EcoR1 at the5′ end of the xln2 promoter in the resulting plasmid, pBR322LXN, wasthen blunted with Klenow and Spe1 linkers (Cat. No. 1086, New EnglandBiolabs) were added to generate pBR322SpXN. A 1.2 kb HindIII fragmentcomprising bp −1399 to −204 of the cbh1 promoter was isolated by HindIIIdigestion of the cbh1 genomic subclone pCB152. This fragment was used toreplace the HindIII fragment comprising bp −1400 to bp −121 of the xln2promoter in the vector pBR322SpXN to generate the plasmid pBR322C/X.

The pBgns2 plasmid was cut with XbaI and NotI and a 4.2 kb fragment,containing the bgl1 coding region lacking the stop codon followed by thecbh2 terminator, was isolated. This fragment was inserted into theplasmid pBR322C/X cut with NheI and NotI (NheI and XbaI have compatibleoverhangs). This cloning resulted in an expression cassette from whichthe mature β-glucosidase lacking the stop codon can be expressed underthe control of the cbh1 promoter and the xln2 secretion signal peptide.This expression cassette plasmid is pC/XBgns and has a unique Kpn1 sitebetween the bgl1 coding region and the cbh2 terminator.

To obtain the cbh1 linker and CBD region, a DNA fragment comprisingbp1665 to bp 1882 of the published cbh1 gene (Shoemaker, et al.) wasamplified by PCR using primers to insert Kpn1 and Spe1 sites at both the5′ end and a Kpn1 site at the 3′ end of the fragment. The 5′ Kpn1 siteis located in order to make a precise fusion between the reading framebetween the bgl1 coding region in pC/XBgns and the reading frame of thecbh1 linker+CBD. The 3′ Kpn1 site is located just after the stop codonof the native cbh1 coding region. This 215 bp PCR product was digestedwith Kpn1 and inserted into the unique Kpn1 site of pC/XBgns, to producethe final expression cassette plasmid, pC/XBg-CBD. As a result of theinsertion of the restriction sites, the final fusion protein expressedby this construct will contain three extra amino acids (Pro-Thr-Ser)between Val713 of the bgl1 coding sequence and the Ile474 of the cbh1coding region.

The E. coli hygromycin phosphotransferase gene (hph) used as aselectable marker for T. reesei was amplified with Pwo polymerase fromthe plasmid pVU1005 (Van den Elzen, Townsend, Lee and Bedbrook, “Achimaeric hygromycin resistance gene as a selectable marker in plantcells”, Plant Mol. Biol. 5: 299-302, 1989). The primers were designed tointroduce Sph1 and Kpn1 sites at the 5′ and 3′ ends of the hph codingregion (bp 211-1236 of the published hph sequence, Gritz and Davies,“Plasmid-encoded hygromycin b resistance: the sequence of hygromycin Bphosphotransferase gene and its expression in Escherichia coli andSaccharomyces cerevisiae” Gene 25: 179-188, 1983), respectively. The PCRproduct was digested with Sph1 and Kpn1 and inserted into thecorresponding sites in the polylinker region of pUC119. The resultingplasmid, pHPT100 was used as the starting plasmid for the constructionof the selection cassette. Two new linker regions were introduced intothis plasmid to facilitate the insertion of the promoter and terminatorfragments required to express the hph gene in a Trichoderma host. AHindIII-XbaI-XhoI-SphI linker was inserted between the HindIII and SphIsites at the 5′ end of the hph sequence and a KpnI-NotI-SacI linkerwhich was inserted between the KpnI and SacI sites at the 3′ end of thehph sequence. This construct was designated as pHPT102. The primers usedto amplify the pgk promoter (Vanhanen, Saloheimo, Ilmen, Knowles andPenttila, “Promoter structure and expression of the 3-phosphoglyceratekinase gene (pgk1) of Trichoderma reesei”, Gene 106: 129-133, 1991) weredesigned to introduce an XhoI site and a SphI site at positions −970 and+1 of the promoter respectively. These sites were subsequently used toinsert the pgk promoter into the XhoI and SphI sites of pHPT102 togenerate pHPT115. A 1.3 kb cbh1 terminator fragment was amplified withPwo polymerase from pCB1Ta using a primer annealing to the 3′untranslated region of cbh1 (bp 1864-1899 of the published cbh1sequence) containing a Kpn1 site at bpl877-1882 and the pUC reverseprimer (Cat. No., 18432-013, Gibco/BRL) which anneals downstream of theEcoR1 site at the 3′ end of the cbh1 terminator in pCB1Ta. The cbh1terminator PCR product was digested with Kpn1 and inserted into theunique Kpn1 site of pHPT115 to generate the selection cassette plasmidpHPT136.

To make the transformation vector, the 5.8 kb expression cassettecomprising a distal 5′ region of the xln2 promoter, bp −1399 to −204 ofthe cbh1 promoter, bp −121 to +99 of the xln2 promoter and secretionsignal peptide, the coding region for the β-glucosidase/CBD fusion andthe cbh2 terminator was isolates from pC/XBg-CBD by digestion with Not1,blunting of the Not1 site with Klenow DNA polymerase, and digestion withSpe1. This 5.8 kb Spe1/Not1 fragment was inserted between the uniqueupstream of the hph selection cassette of pHPT136 which had beendigested with Xho1, blunted with Klenow DNA polymerase and digested withXba1 (Spe1 and Xba1 have compatible overhangs). The final transformationvector, pC/XBg-CBD-TV, was linearized at the unique Not1 site at the 3′end of the cbh1 terminator in the hph selection cassette and introducedas a linear vector into T. reesei BTR213 via microprojectile bombardmentas described below.

5.3 Transformation of T. reesei BTR213 via Microprojectile Bombardment

The Biolistic PDS-1000/He system (BioRad; E.I. DuPont de Nemours andCompany) was used to transform spores of T. reesei strain BTR213 and allprocedures were performed as recommended by the manufacturer. M-10tungsten particles (median diameter of 0.7 um) were used asmicrocarriers. The following parameters were used in the optimization ofthe transformation: a rupture pressure of 1100 psi, a helium pressure of29 mm Hg, a gap distance of 0.95 cm, a macrocarrier travel distance of16 mm, and a target distance of 9 cm. Plates were prepared with 1×10⁶spores on Potato Dextrose Agar media (PDA). Bombarded plates wereincubated at 28° C. Four hours post-bombardment, spores are subjected toprimary selection by the overlaying of selective PDA media supplementedwith 40 units/mL of HygB. The bombardment plates are incubated at 28° C.Transformants can be observed after 3-6 days growth; however, furtherincubation is necessary to achieve sporulation.

After sporulation has occurred, a secondary selection process isperformed to isolate individual transformants. Spores are collected fromthe plate with an inoculating loop and re-suspended in sterile water.This suspension is then filtered through a sterile syringe plugged withglass microfibers. This allows the passage of spores while retainingunwanted mycelia. A determination of the concentration of spores in thissuspension is required and subsequent dilutions are plated onto PDAplates supplemented with 0.75% Oxgall (Difco) and HygB (20 units/mL) toobtain 20-50 spores per plate. The Oxgall acts as a colony restrictor,thereby allowing the isolation of individual colonies on these secondaryselection plates. Isolated colonies can be observed after 2-3 days.

5.4 Production of β-glucosidase in Liquid Cultures

Individual colonies of Trichoderma are transferred to PDA plates for thepropagation of each culture. Sporulation is necessary for the uniforminoculation of shake flasks which are used in testing the ability of theculture to produce the β-glucosidase and cellulase. The culture media iscomposed of the following:

TABLE 2 Components of the culture media Component Concentration(NH₄)₂SO₄ 6.35 g/L KH₂PO₄ 4.00 g/L MgSO₄•7H₂O 2.02 g/L CaCl₂•2H₂O 0.53g/L Corn Steep Liquor 6.25 g/L CaCO₃ 10.00 g/L Carbon sources** 5-10 g/LTrace elements* 1 mL/L *Trace elements solution contains 5 g/LFeSO₄•7H₂O; 1.6 g/L MnSO₄•H₂O; 1.4 g/L ZnSO₄•7H₂O. **5 g/L glucose plus10 g/L Solka floc (when the cbh1 or other cellulase promoter is used),10 g/L xylan (when the xln2 promoter is used) or other carbon sourcecompatible with the promoter directing the expression of theβ-glucosidase. The carbon source can be sterilized separately as anaqueous solution at pH 2 to 7 and added to the remaining media.

The liquid volume per 1-liter flask is 150 mL, the initial pH is 5.5 andeach flask is sterilized by steam autoclave for 30 minutes at 121° C.prior to inoculation.

For both native and transformed cells, spores are isolated from the PDAplates as described in Section 5.3 above and 1−2×10⁶ spores are used toinoculate each flask. The flasks are shaken at 200 rpm at a temperatureof 28° C. for a period of 6 days. The filtrate containing the secretedprotein was collected by filtration through GF/A glass microfibrefilters (Whatman). The protein concentration is determined using theBio-Rad Protein Assay (Cat. No. 500-0001) using Trichoderma cellulase asa standard. β-glucosidase activity is determined as described in Ghose,1987.

5.5 Production of β-glucosidase by T. reesei Strains BTR213 and 1059Ausing Solka Floc Carbon Source

The native strain BTR213 and the transformed strain from this host 1059Awere cultured using the procedures of Example 5D with 10 g/L Solka flocand 5 g/L glucose as carbon sources. The results are shown in Table 2.

The native strain produced 0.19 IU of β-glucosidase per mg protein.

The transformant 1059A expressing the β-glucosidase/CBD fusion from thecbh1 promoter and xln2 secretion signal produced 7.6 IU/mg ofβ-glucosidase. This represents a 40-fold increase over the nativestrain, which represents the vast majority of the β-glucosidase.

TABLE 3 Production of β-glucosidase activity from T. reesei strainsBTR213 and 1059A in 150 mL flask cultures Secretion β-glucosidase StrainPromoter signal β-glucosidase (IU/mg) RutC30 bgl1 bgl1 Native 0.14RC-302 cbh1 xln2 β-G/CBD 19 fusion

1. A process for the enzymatic hydrolysis of cellulose to produce ahydrolysis product comprising glucose from a pretreated lignocellulosicfeedstock, the process comprising: (i) providing an aqueous slurry ofthe pretreated lignocellulosic feedstock, said aqueous slurry comprisingfiber solids and an aqueous phase; (ii) hydrolyzing the aqueous slurrywith cellulase enzymes that bind to the fiber solids and one or morethan one β-glucosidase enzyme having a cellulose binding domain thatbinds the fiber solids, said hydrolyzing being carried out in asolids-retaining hydrolysis reactor to produce said hydrolysis productcomprising glucose; and (iii) withdrawing unhydrolyzed solids and theaqueous phase, which comprises hydrolysis product, from thesolids-retaining hydrolysis reactor, wherein the unhydrolyzed fibersolids are retained in the solids-retaining hydrolysis reactor for amean time of about 6 hours to about 148 hours longer than the aqueousphase.
 2. The process according to claim 1, wherein the cellulaseenzymes comprise at least one cellobiohydrolase enzyme selected from thegroup consisting of CBHI and CBHII cellulase enzymes, and at least oneendoglucanase enzyme selected from the group consisting of EGI, EGII,EGIII, EGIV, EGV and EGVI cellulase enzymes.
 3. The process according toclaim 1, wherein, in the step of providing (step (i)), the aqueousslurry has a suspended or undissolved fiber solids content of about 3%to about 30% (w/w).
 4. The process according to claim 1, furthercomprising a step of separating the hydrolyzed solids from thehydrolysis product comprising glucose to produce separated solids and anaqueous solution comprising glucose.
 5. The process according to claim4, wherein the unhydrolyzed solids are separated from the hydrolysisproduct by microfiltration, centrifugation, vacuum filtration orpressure filtration.
 6. The process according to claim 5, wherein theunhydrolyzed solids are separated from the hydrolysis product bymicrofiltration.
 7. The process according to claim 1, wherein thesolids-retaining hydrolysis reactor is part of a hydrolysis systemcomprising two or more hydrolysis reactors, and the hydrolysis reactorsare operated in series, in parallel, or a combination thereof.
 8. Theprocess according to claim 7, wherein the hydrolysis system comprisesone or more than one hydrolysis reactor selected from the groupconsisting of an agitated tank, an unmixed tank, an agitated tower andan unmixed tower.
 9. The process according to claim 8, wherein theagitated tower is an upflow tower.
 10. The process according to claim 8,wherein the unmixed tower is an upflow tower.
 11. The process accordingto claim 1, wherein the aqueous slurry is concentrated prior to the stepof hydrolyzing (step (ii)).
 12. The process according to claim 1,wherein the process is a batch process.
 13. The process according toclaim 1, wherein the process is a continuous process with continuousfeeding of the aqueous slurry and continuous withdrawal of theunhydrolyzed solids and the aqueous phase comprising the hydrolysisproduct.
 14. The process according to claim 1, wherein, in the step ofhydrolyzing (step (ii)), about 70% to about 100% of cellulose in thefiber solids is converted to glucose.
 15. The process according to claim1, further comprising a step of separating the unhydrolyzed solids fromthe hydrolysis product comprising glucose to produce separated solidsand a first aqueous solution comprising glucose, re-suspending theseparated solids in an aqueous solution to produce a re-suspendedslurry, continuing the hydrolysis of the re-suspended slurry in a secondhydrolysis and obtaining a second aqueous solution comprising glucosefrom said second hydrolysis.
 16. The process according to claim 15,wherein the re-suspended slurry has a solids concentration of about 10%to about 15% (w/w).
 17. The process according to claim 15, wherein thestep of continuing the hydrolysis is carried out for about 12 to about200 hours.
 18. The process according to claim 15, further comprisingfermenting the first aqueous solution comprising glucose, the secondaqueous solution comprising glucose, or a combination thereof, toproduce a fermentation broth comprising ethanol.
 19. The processaccording to claim 1, wherein, in the step of providing (step (i)), thepretreated lignocellulosic feedstock is obtained from wheat straw, oatstraw, barley straw, corn stover, soybean stover, canola straw, ricestraw, sugar cane, bagasse, switch grass, reed canary grass, cord grass,or miscanthus.
 20. The process according to claim 1, wherein, in thestep of hydrolyzing (step (ii)), the cellulase enzymes are added at adosage of about 1.0 to about 40.0 IU per gram of cellulose.
 21. Theprocess according to claim 1, wherein, in the step of hydrolyzing (step(ii)), the one or more than one β-glucosidase enzyme is added at adosage of 35 to about 200 IU per gram of cellulose.
 22. The processaccording to claim 1, wherein, in the step of hydrolyzing (step (ii)),the cellulase enzymes are obtained from Aspergillus, Humicola,Trichoderma, Bacillus, Thermobifida, or a combination thereof.
 23. Theprocess according to claim 1, wherein, in the step of hydrolyzing (step(ii)), the β-glucosidase enzyme is obtained from Aspergillus, Humicola,Trichoderma, Bacillus, Thermobifida, or a combination thereof.
 24. Theprocess according to claim 23, wherein the β-glucosidase enzyme isobtained from Aspergillus or Trichoderma.
 25. The process according toclaim 1, wherein the step of hydrolyzing (step (ii)) is carried out fora total residence time of the aqueous phase of about 12 to about 200hours.
 26. The process according to claim 1, wherein about 75% to about100% (w/w) of the total cellulase enzymes present bind to the fibersolids in the step of hydrolyzing (step (ii)).